Compositions and methods involved in bone growth

ABSTRACT

Disclosed are compositions and methods related to osteoblasts, osteoclasts, and osteoclast lacunae.

This application claims priority to U.S. Provisional Application No.60/323,987 filed on Sep. 20, 2001, entitled “Compositions and methodsinvolved in bone growth,” which application is herein incorporated byreference in its entirety.

I. ACKNOWLEDGEMENT

This invention was made with government support under federal grants RO1DE 12011 and ROI ES 08121, awarded by the NIH. The Government hascertain rights to this invention.

II. BACKGROUND

The concept of a coupled communication between osteoblastic boneformation and osteoclastic bone resorption was first described in humansby Harris and Heaney in 1969 (Harris W H. Heaney R. P., New EnglandJournal of Medicine. 280(5):253-9 contd, 1969; Harris W H. Heaney R P.,New England Journal of Medicine. 280(6):303-11 concd 1969). They showedthat in patients with high rates of resorption there was acorrespondingly high rate of bone formation and concluded that themaintenance of a steady state skeletal mass required that osteoclasticbone resorption be matched in amount by osteoblastic bone formation.They based their investigations on conceptual principles put forward byFrost (Epker B N. Frost H M., Henry Ford Hospital Medical Journal.16(1):29-39, 1968; Villanueva A R. et al., Clinical Orthopaedics &Related Research. 49:135-50, 1966; Frost H M., Journal of Bone & JointSurgery—American Volume. 48(6):1192-203, 1966) and Parfit (Parfitt A M.,Clinical Obstetrics & Gynecology. 30(4):789-811, 1987; Parfitt A M.,Metabolism: Clinical & Experimental. 25(8):909-55, 1976). William Harrisand Robert Heaney coined the term “coupling.”

Coupling was defined at a molecular and cellular level by Howard,Baylink and others (Howard G A. et al., Progress in Clinical &Biological Research. 101:259-74, 1982; Howard G A. et al., Proceedingsof the National Academy of Sciences of the United States of America.78(5):3204-8, 1981; Baylink D. et al., Advances in Experimental Medicine& (Biology. 151:409-21, 1982.) as a release of growth factors from boneduring resorption leading to the subsequent activation anddifferentiation of osteoblasts for the process of bone formation. Theypresented evidence for a “coupling factor” that could be recovered inthe medium of bone undergoing resorption. Coupling factor turned out tobe a number of molecules including the IGF's, TGFβ's and BMP's.

Therefore, both systemic hormones such as PTH and local factors such asthe TGFβ's, BMP's and IGF's are molecules with the potential tostimulate both osteoblast number and osteoblast differentiation.However, what has not been universally appreciated as part of theremodeling process is that bone formation occurs on the immediatesurface of the resorptive event. That is, growth factor diffusion from aresorption site occurs with a larger radius than is encompassed by theactual site of bone formation, yet bone is deposited at the site of boneresorption. This argues that there must be a site-specific localizationto the formation process. Teleologically, this makes sense, since ifbone were deposited at sites other than those undergoing resorption, itmight lead to alteration of trabecular architecture and contribute tothe formation of structurally unsound bone.

Osteoclastic bone resorption depends on the formation of adhesive bondsbetween the cell and the bone surface as well as the formation of twocritical ultrastructural features; the ruffled boarder and sealing (orclear) zone. At the ruffled border the cell actively pumps large numbersof protons into the space between the cell and bone. pH is decreased tothe range of approximately 5.0 to 6.0 and the hydroxyapatite becomessoluble. Secretion of lysosomal enzymes and other acidic hydrolases intothis space begins the process of collagen and non-collagen proteinbreakdown. In many ways the space between the osteoclast and the bonesurface becomes an extracellular lysosome characterized by high levelsof lysosomal enzymes and a low pH. In fact, the brush border membrane ofthe osteoclast has been shown to contain high levels of themannose-6-phosphate receptor (A1 Kawas S. et al., Calcified TissueInternational. 59(3):192-9, 1996; Blair H C. et al., ClinicalOrthopaedics & Related Research. (294):7-22, 1993 September; Baron R. etal., Journal of Cell Science. 97 (Pt 3):439-47, 1990; Baron R. et al.,Journal of Cell Biology. 106(6):1863-72, 1988 June). This receptor isresponsible for trafficking lysosomal enzymes to a lysosome. Whenpresent on osteoclast (or ameloblast) brush border membranes itpresumably can direct these enzymes to the active resorbing surface.These osteoclast enzymes remain firmly attached to the resorptionsurface (Xia L. et al., Biological Chemistry. 380(6):679-87, 1999 June;Romano P R. et al., Journal of Periodontal Research. 32(1 Pt 2):143-7,1997).

Mannose-6-phosphate moieties present on lysosomal enzymes have theability to activate the mannose-6-phosphate receptor. Since themannose-6-phosphate receptor shares identity with the IGF-II receptor,activation of this receptor by lysosomal enzymes could induce a growthfactor like effect (Ishibe, M., et al., J. Clin. Endocrinol. Metab. 73:785-792 (1991); Ishibe, M., et al., Endocrine Research. 17: 357-366(1991); Martinez, D. A., et al., (1995) J. Cellular Biochemistry 59:246-257; Ishibe, M., et al., Cal. Tissue Intl. 63:36-38 (1998).).

Thus, there exists a need for identification of the molecules involvedin mediating the osteoblast/osteoclast/osteoclast lacuna interactionsand signalings. Disclosed herein are compositions and methods that areinvolved in these interactions and can mediate the boneresorptive/formative event.

III. SUMMARY

Disclosed are compositions and methods involved in interactions withosteoblasts, osteoclasts, and/or osteoclast lacuna.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments and togetherwith the description, serve to explain the principles of the disclosedcompositions and methods.

FIG. 1 shows a diagram of bone resorption and formation.

FIG. 2. shows a bio-panning strategy for use with phage display. Both arandom and an osteoblast cDNA phage display library have been utilized.This figure depicts a random library in M13. The “bait” is immobilizedTRAP on a tissue culture dish. After three rounds of panning, elutionand amplification, the remaining adherent phage are sequenced.

FIG. 3A shows osteoblast binding as a function of resorption area. Bonewafers with different amounts of osteoclast lacunae were used todemonstrate that more osteoblasts adhere to wafers with more lacunae.FIG. 3B shows osteoblasts cultured on wafers with pitted osteoclastlacunae are stimulated to produce more alkaline phosphatase andproliferate at a slightly greater rate. Treatment of the wafers with aendoglycosidase H blocks some of the effect.

FIG. 4 shows the effect of TRAP on osteoblast proliferation, collagensynthesis and alkaline phosphatase activity. TRAP (purified uteroferrindonated by Dr. M. Roberts) was exposed to osteoblasts in culture in adose dependent fashion. There was a modest stimulation of proliferationand marked stimulation of differentiation as noted by collagen andalkaline phosphatase increases. Treatment of the TRAP with a glycosidaseeliminated the effect on alkaline phosphatase. N=4-6. “*” statisticallysignificant at p 0.05.

FIG. 5 shows ELISA assay for the five phage clones with highestconsensus frequency from Table 4. TRAP, immobilized in 96 well culturewells, was used as the substrate to demonstrate the affinity of phage.All of these phage demonstrated at least a 40 fold higher binding thanwild type (WT) phage. Clone 5 phage (dark bar) was used for furtheranalyses. Non of the phage demonstrated a statistically significantdifference from each other, however, all were different from control.

FIG. 6 shows a Scatchard-type analysis for Clone 5 phage binding toTRAP. Immobilized TRAP was used as a substrate and phage binding wasdetermined by measuring the titer of phage in the unbound fraction. A Kdin the sub-nanomolar range was obtained

FIG. 7 shows a principle of a Far-Western. Phage are used as the probesto identify specific proteins separated by gel electrophoresis. Thephage are identified with a HRP-conjugated antibody directed against aphage coat protein.

FIG. 8 shows inhibition of osteoblast binding. A synthetic peptidecorresponding to the Clone 5 sequence effectively competes withosteoblast binding to pitted bone wafers. Osteoblasts prelabelled with3H-leucine were used to measure cell affinity for cortical bone wafersin the presence and absence of resorption lacunae. As shown in FIG. 3osteoblasts bind with higher affinity to pitted wafers. However, a Clone5 peptide can inhibit the binding at as little as 10 pM. N=8.

FIG. 9 shows effect of TRAP on activation of a TGFβ reporter construct(P3TPlux). P3TP lux was transfected into osteoblasts and they were thenexposed to TRAP or TGFβ Panel A shows that TRAP can activate P3TP lux ina dose dependent fashion. Panel B shows that, as expected, TGFβ canstrongly activate P3TP lux but that addition of TRAP augments the TGFβeffect. Note the difference in scales. N=4-6. All data points arestatistically significantly different from the first bar in theirrespective graph.

FIG. 10 shows measuring cell affinity for a substrate. The horizontalaxis is the touch number and vertical axis is the cumulative percent oftouches that lead to an adhesive event. Each point represents the meanof at least 9 cells. Error bars are omitted so as not to clutter thefigure.

FIG. 11 shows TGFβ activation pathways. Three main regulatory pathwaysconverging on two transactivating transcription factors have beendescribed for TGFβ activation. The pathways have been highlighted andfrom left to right they are: the JNK pathway, the p38 pathway and theSmad pathway.

FIG. 12 shows a mammalian two-hybrid assay for TRAP and glypican 4(GPC4) interaction. TRAP was expressed as a fusion protein with Gal4 BD(Gal4-BD-TRAP). GPC4 was expressed as a fusion protein with VP16AD(VP-16-AD-GPC4). When both fusion constructs were co-transfected intoMG-63 cells, the association of TRAP with GPC4 allowed for promoteractivation and polymerase activity to proceed for the transcription andtranslation of luciferase. Control transfections with single constructsof Gal4-BD-TRAP and VP-16-AD-GPC4 did not stimulate luciferaseexpression. Transfection with a VP-16-AD-GPC4 construct deficient in the12 amino acids of Clone 5 (VP-16-AD-GPC412) also could not stimulateluciferase expression. Moreover, and most importantly, co-transfectionof Gal4-BD-TRAP with VP-16-AD-GPC412 did not stimulate luciferaseactivity.

FIG. 13 shows clone 5 peptide interferes with osteoblast binding toosteoclast lacunae. Osteoblasts bind in greater number to waferscontaining osteoclasts pits. A synthetic peptide corresponding to theClone 5 sequence can interfere with this osteoblast binding in a dosedependent fashion. “†” indicates a statistically significant differencebetween osteoblast binding on osteoclast pitted wafers vs. un-pittedwafers (p 0.005). “*” indicates a statistically significant differencebetween wafers treated with the Clone 5 peptide and control pittedwafers (to p levels of at least 0.05). Each value is the mean±one SEM;N=6.

FIG. 14 shows the effect of TRAP and TGFβ on osteoblast phenotypicmarkers. The qualitative pattern of stimulation by TRAP and TGFβ formarkers of osteoblast differentiation is similar. The concentration ofTRAP was 10 μg/ml and for TGFβ was 2 ng/ml for all determinations.Alkaline phosphatase (Alk. Phos.) was measured in a direct biochemicalassay. Runx2 and osteoprotegerin (OPG) were quantified in an ELISA assayusing commercially available antibodies. Collagen was measured bydetermining the amount of collagenase-digestible protein present inpre-labeled osteoblasts (34). All data are expressed as a percent of thecontrol, untreated cells. All data points are the mean±one SEM. * p0.05. ** p 0.01

FIG. 15 shows the specificity and affinity of TRAP binding to TRIP-1.FIG. 15A demonstrates that wild type phage (containing no osteoblastproteins) have little affinity for TRAP that has been immobilized in aculture dish. TRIP-1-containing phage show a dose dependent increase inTRAP binding. Phage titer is expressed as particles/ml. Phage bindingwas detected with HRP-anti T7 phage antibody in a sandwich ELISA assay.All TRIP-1 phage data points are the mean±one SEM. ** p 0.01. FIG. 15Bdemonstrates the specificity of TRIP-1 binding to TRAP. Human TRIP-1 wassynthesized as a GST fusion protein. The GST vector alone served as acontrol protein. Different amounts of TRAP and other proteins (i.e.bovine serum albumin, BSA) were incubated with the GST-TRIP-1 fusionprotein and the molecules associating with the GST-TRIP-1 were extractedby exposure to glutathione beads. The “pulled-down” proteins wereseparated on a denaturing gel and the level of TRAP was measured withFar Western analysis. Lane A shows that GST has no affinity for TRAP.Lane B shows that TRIP-1 does not bind to any molecules in BSA thatwould be detected in the Far Western. Lanes C and D show that increasingamounts of TRAP can be captured by the GST-TRIP-1 in a dose-dependentfashion. Lane E is a control lane loaded with TRAP to demonstrate thedetection of this system.

FIG. 16 shows a two hybrid demonstration of TRAP association withTRIP-1. A mammalian two-hybrid system was utilized to demonstrate anassociation between TRAP and TRIP-1. 293T cells, when individuallytransfected with a Gal4-DBD-TRIP-1 or VP-16-AD-TRAP fusion protein,showed no activation of the luciferase reporter. However,co-transfection of the cells with both constructs allowed forinteraction of TRAP with TRIP-1. This association permitted the assemblyof the transcription machinery for luciferase expression. Substitutionof an anti-sense form of TRIP-1 as the Gal4-DBD fusion protein, asexpected, did not show any association with TRAP. All data are the meanof four determinations±SEM. ** p 0.01 as compared Gal4-BD-TRAP.

FIG. 17 shows TRAP and TGFβ activate the Smad signaling pathway. P3TPLuxis a Smad 2, 3 sensitive reporter. Transfection of this reporter intoeither of the osteoblast cell lines, SaOS2 or MG-63, and exposure of thecells to TRAP (10 μg/ml) or TGFβ (5 ng/ml) causes an activation of theSmad pathway. The effect of TRAP plus TGFβ are additive. Co-transfectionof the cells with a dominant negative TGFβ type II receptor blocks allTRAP and TGFβ signaling. Each bar is the mean of at least fourdeterminations±SEM. * p 0.05 and ** p 0.01 as compared to control. Thedata for MG-63 cells are shown. The data for the SaOS2 cells are thesame (data not shown).

FIG. 18 shows TRAP and TGFβ signaling in a Smad4 deficient cell line.SW408 cells are deficient in Smad4, a requisite co-factor for Smad2 and3 signaling. The data in this figure demonstrate that neither TRAP norTGFβ (nor the combination of factors) can activate the Smad signalingpathway in SW408 cells. However, when the cells are transfected withboth Smad4 and TRIP-1, restoration of Smad signaling can occur. Allvalues are the mean±SEM. * p 0.05 and ** p 0.01 as compared to control.

FIG. 19 shows a demonstration of a TRIP-1, TGFβ type II receptor, Smad2complex with TRAP. 293T cells were transfected with TGFβRII. Some ofthese cells were then co-transfected with a “His”-tagged TRIP-1(His-TRIP-1). In lane A, in which both the receptor and TRIP-1 werepresent, but GST-TRAP was not added, no parts of the complex could be“pulled down” with glutathione coated beads. The same was true ifHis-TRIP-1 was not transfected into the cells. However, if the receptor,TRIP-1 and GST-TRAP were all present, a protein complex composed of thereceptor, TRIP-1 and Smad 2 could be extracted with glutathione coatedbeads. These data indicate that TRAP, TRIP-1, the TGFβRII and Smad2 canbe found is close association with each other.

FIG. 20 shows DNA laddering, a hallmark of apoptosis, also occurs in theROS17/2.8 cell line after exposure to TRAP.

FIG. 21A shows that early passage rat osteoblasts (day 3) (D3 ROB) andMC3T3-E1 cells have very low levels of collagenase III. This levelincreases as the rat osteoblasts mature (day 14) (D14 ROB) and reaches amaximum in ROS 17/2.8 cells. FIG. 21B demonstrates an increase in thesenescence-associated, galactosidase in day 14 osteoblasts, with andwithout the addition of the differentiating agent β-glycerol phosphate.

FIG. 22 shows an examination of apoptotic DNA fragmentation in ROS17/2.8 cells.

FIG. 23 shows that TRAP induces an increase in both cleaved caspase 3and cleaved PARP in ROS 17/2.8 cells.

FIG. 24 shows the effect of TRAP on cleavage of PARP by day 14 cells butnot day 3 cells.

FIG. 25 shows cleavage of caspase 3 in day 14 cells treated with TRAP.

FIG. 26 shows ELISA assays that determine the levels of OPG in primaryosteoblast cells.

V. DETAILED DESCRIPTION

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that theyare not limited to specific synthetic methods, specific recombinantbiotechnology methods unless otherwise specified, or to particularreagents unless otherwise specified, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

A. Definitions

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

“Probes” are molecules capable of interacting with a target nucleicacid, typically in a sequence specific manner, for example throughhybridization. The hybridization of nucleic acids is well understood inthe art and discussed herein. Typically a probe can be made from anycombination of nucleotides or nucleotide derivatives or analogs ornucleotide substitutions available in the art.

“Primers” are a subset of probes which are capable of supporting sometype of enzymatic manipulation and which can hybridize with a targetnucleic acid such that the enzymatic manipulation can occur. A primercan be made from any combination of nucleotides or nucleotidederivatives or analogs or nucleotide substitutions available in the artwhich do not interfere with the enzymatic manipulation.

Disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves and to be usedwithin the methods disclosed herein. These and other materials aredisclosed herein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collective permutationof these compounds may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a particular TRIP isdisclosed and discussed and a number of modifications that can be madeto a number of molecules including the TRIP are discussed, specificallycontemplated is each and every combination and permutation of TRIP andthe modifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the disclosed compositions. Thus, if there are a variety ofadditional steps that can be performed it is understood that each ofthese additional steps can be performed with any specific embodiment orcombination of embodiments of the disclosed methods.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art. The references disclosed are alsoindividually and specifically incorporated by reference herein for thematerial contained in them that is discussed in the sentence in whichthe reference is relied upon.

B. Compostions and methods

FIG. 1, shows a diagram of the process that occurs during boneresorption and formation. Bone resorption occurs when osteoclasts begin,through lysosomal enzymes and pH regulation of the extracellular spacebetween the bone and the osteoclasts, to degrade the bone at thejunction of the osteoclasts and the bone. This produces an osteoclastlacunae which in turn is specifically recognized by osteoblasts.Disclosed herein are compositions and methods that are drawn to thisrecognition event between osteoblasts and osteoclast lacunae. Once theosteoblasts recognize the osteoclast lacunae, bone formation can begin,through differentiation and proliferation of osteoblasts. This processof differentiation and proliferation occurs at the site of resorption,the osteoclast lacunae and is mediated by this region. Compositions andmethods are disclosed which are drawn to and involved in this process ofdifferentiation and proliferation. Thus, disclosed are compositions andmethods, which are related to bone resorption and bone formation.

Disclosed are compositions and methods related to osteoblasts,osteoclasts, and osteoclast lacunae. Disclosed are peptides thatinteract with TRAP and with osteoclast lacunae. One of these peptideshas substantial identity to a region of GPC4 (SEQ ID NO:38), which is apeptide that is expressed in osteoblasts, and full length GPC4, asdisclosed herein, interacts with TRAP. Thus, compositions and methodsthat enhance or inhibit the interaction of GPC4 and TRAP, and thusenhance or inhibit the interactions of osteoblasts with osteoclastsand/or osteoclast lacuna, are disclosed.

Furthermore, it is disclosed that TRAP (SEQ ID NO:42) interacts withTRIP (TGFβ receptor interacting protein) (SEQ ID NO:40), which is aG-protein coupled cell signaling protein. It is shown that TRIP isexpressed in osteoblasts. Thus, compositions and methods for affectingthe interactions between TRAP and TRIP are disclosed. These compositionsand methods in turn ultimately effect the bone formation propertiescontrolled by TRAP and the interaction between osteoblasts andosteoclast lacuna.

It is also disclosed that TRAP can activate the Smad3/Smad4 TGFβsignaling pathway as well as that TRAP can stimulate osteoblastdifferentiation. Thus, disclosed are compositions and methods thataffect TGFβ signaling pathways, such as the Smad3/Smad4 pathway andcompositions and methods that affect the osteoblast differentiation, forexample, through a TGFβ pathway.

It is also disclosed that TRAP can induce apoptosis of mature or endstage osteoblast cells. This apoptosis is mediated through TRIP utilizesa Ras/Raf pathway. Thus, disclosed are compositions and methods thataffect the TRAP signaling pathways, involving apoptosis, such ascompositions and methods that interrupt signaling between TRAP andRas/Raf. Also disclosed are compositions and methods that affect theosteoblast apoptosis, for example, through a Ras/Raf pathway.

1. Apoptosis

Osteocyte apoptosis has been speculated to be a signal for boneresorption by osteoclasts in growing bone, and increased activationfrequency in estrogen-deficiency osteoporosis. Osteocytes are theterminal differentiation stage of osteoblasts. Osteoclasts can induceosteocyte apoptosis by many singaling pathways. FasL, displayed on thesureface of osteoclast, has been shown to directly bind to a receptor,Fas, found on the osteoblasts and osteocytes. Activation of the Fasreceptor leads to apoptosis.

Osteoblast apoptosis occurs at sites of local remodeling and fracturerepair in adult bone (Burger E H, Klein-Nulend J., Faseb J 13Suppl:S101-12, 1999; Grzesik W J, Robey P G, J Bone Miner Res 9:487-96,1994; Mundy G R, et al., Calcif Tissue Int 34:542-6, 1982; Schwartz Z.,et al., Adv Dent Res 13:38-48, 1999).

Only a 30-50% transient increase osteoblast numbers will become theirterminal differentiation stage (lining cell and osteocyte). Uponcompletion of new bone formation the cellular excess must be resolved bythe controlled removal of the surplus cells. Alternative mechanisms forelimination of excess osteoblasts include differentiation, emigration,or cell death. Evidence for the latter course has been provided by theobservation of local and selective cell death in the mineralizing rodentcalvarium (Spanner M, et al., Bone 17:161-5, 1995).

In many tissues, apoptosis is the major mechanism of cell deathresponsible for modulating cell population size on a local basis(Parfitt A M, Bone Histomorphometry: Techniques andInterpretation.:143-223, (1983)) and is a likely candidate for thephysiological regulation of osteoblast cell numbers in adult bone. Thegrowth factors and cytokines, which can modulate and induce apoptosis inosteoblasts are thought to be TNF-α (Hock J M, et al., J Bone Miner Res16:975-84, 2001). FasL (.Abe Y, et al., J Lab Clin Med 136:344-54,(2000)), IL-6 and TGF-β may induce apoptosis in osteoblastic cells.

Disclosed herein TRAP protein was added to different bone cell linesthat represented either a progenitor or early stage osteoblast as wellas a mature or end stage osteoblast. TRAP induced differentiation inearly stage osteoblasts and osteoblast progenitors and caused apoptosisin mature or end stage osteoblasts. We also attempted to characterizethe TRAP signaling pathway involved in this apoptosis-inducing effect.The traditional Ras and Raf signaling pathways were involved in the TRAPmediated apoptosis.

The cytological stages of apoptosis are characterized by rapidcondensation of chromtin and budding of the cell membrane into enclosedapoptotic bodies containing well preserved organelles. A characteristicbiochemical feature of the chromatin condensation is a double break ofnuclear DNA at the linker regions between nucleosomal fragments. Thisnucleosomal fragmentation of DNA results from the activation ofnucleases in cells. One such nuclease, DNA fragmentation factor (DFF, acaspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD), iscapable of inducing DNA fragmentation and chromatin condensation aftercleavage by caspase-3.

Osteoclastic bone resorption can only be initiated on a bare mineralsurface. However, virtually all bone surfaces are covered with a layerof stromal cells. These quiescent stromal cells are derived from maturedand scenescent osteoblasts that have completed a phase of formation inthe past. The question remains; how does the approaching osteoclastclear these cells prior to attachment and initiation of bone formation.Disclosed herein it is consistent with the early secretion of enzymes(i.e. TRAP) in the vicinity of the stromal cells which cause the matureosteoblasts to undergo apoptosis which exposes the mineral surface forthe osteoclast.

In vitro, late stage can be defined as the number of days in culture. 14days of culture would represent late stage and 3 days would representearly stage. Late and early stage characteristics of the cells, asdiscussed herein, can also be used to determine what stage the cells arein. For example, biochemical or morphological markers as discussedherein can be used to identify the stage of the bone cell. In vivo, i.e.in an animal or human, late stage can be defined by the morphology ofthe cells. A late stage osteoblasts is flattened against the bonesurface, elongated and showing no metabolic activity. An early stageosteoblast is plump, polygonal and a produces protein.

Biochemical confirmation of the effect of TRAP on osteoblast apoptosiscan be obtained by assaying levels of caspase-3 and poly ADP-ribosepolymerase (PARP) in these cells lines. Caspase-3 is an enzyme that is akey member of the apoptotic cascade. However, most cells have aconstitutive amount of this enzyme. It is not until the caspase(s) arecleaved that they become activated. Any elevation of caspase 3 over thelevels in early stage cells would be indication of the onset ofapoptosis. It is a relative measure, in that the level of caspase 3 inthe cell is compared to a control, early stage cell, such as a 3 day oldcell.

Thus, another indicator of a cells progression through apoptosis is ameasurement of its cleaved caspase 3 levels. Similarly, poly ADP-ribosepolymerase is a substrate for active caspase 3 and increased levels ofcleave PARP are indicative of an active caspase.

The stage of maturation of the osteoblast cell lines can also bedetermined by measuring collagenase III levels and levels of thesenescence-associated b-galactosidase enzyme. Collagenase III (MMP-13)is marker for late stage osteoblast differentiation. It is induced byhormones and factors that enhance osteoblast development. Thus, presenceof the collagenase III MMP-13) marker indicates late stage or matureosteoblasts.

ROS 17/2.8 osteosarcoma cells represent a highly differentiated stage ofthe osteoblastic lineage despite their transformed phenotype. They canexpress both high level of osteocalcin, alkaline phosphatase and the E11antigen defining the osteoblast-osteocyte transition. Latedifferentiation stage primary osteoblastic cells can be made by usingthe in vitro cell senescence method (Ishibashi O, et al., Calcif TissueInt 68:109-16, (2001)).

These aged cells are similar to other diploid cell models widely used inresearch on cellular aging (Bronckers A L, et al., J Bone Miner Res11:1281-91, (1996)) and they exhibit phenotypic characteristics of invitro cellular senescence, including a limited proliferative capacity inculture, a progressive decline in the rate of macromolecular synthesisand a dramatic change in morphology (an increase in cell size andspreading, and a changed organization of the cytoskeleton) (Howard G A,et al., Proc Natl Acad Sci USA 78:3204-8, (1981)).

Also, altered gene expression, higher levels of cytoplasmic neutralβ-galactosidase activity, as well as a relative increase in geneexpression of the osteoblast-specific collagenase III was demonstratedduring aging of these cell lines in culture.

In the results disclosed herein, ROS 17/2.8 cells were incubated with0.1% DMSO to scavenge the intracellular reactive oxygen species and thenincubated with TRAP (5 ug/mL). DMSO, at this low concentration, itselfwon't cause any toxic effect on cells but can scavenge most of reactiveoxygen species. However, this treatment did not protect cells fromapoptosis induced by TRAP. Therefore, the TRAP-induced cell apoptosis isnot due to a reactive oxygen species effect.

Disclosed herein the late stage (D14) of osteoblasts have lessOsteoprotegrin (OPG) production when the cells have been treated withTRAP proteins and TGF-β. OPG is the deccoy receptor for the RNAKL andstop the osteoclastogenesis. RANKL and OPG has been proposed for theregulation of osteoclast differentiation function. Both OPG and RANKLcan themselves also be regulated by TGF-β (MacDonald R G, Science239:1134-7, (1988)). TRAP proteins also have the same effect of that ofTGF-β to stimulate the osteoblast secreting OPG. The aged osteoblastshave less response to TRAP proteins and TGF-β. This lower the secretionamount of OPG the less the protection afforded from the osteoclast.

Certain branches of the TGF-β signaling pathway can be blocked bytransfecting dominated negative form of Smad 2, 3, 4 and smurf 1 and 2(smad specific ubquitin ligase) separately. Transfection of thesedominant negative molecules did not affect the TRAP-induced apoptosis ina ROS 17/2.8 cell. There are also SMAD independent TGF-β signalingpathways also existed (Wrana J L., Cell 100:189-92, (2000), MacDonald RG, Science 239:1134-7, (1988)).

For instance, TGF-β rapidly activates Rho family guanosinetriphophatases (GTPases); MAPKs, including ERKs, p38, and JNKs throughtheir upstream kinase activators such as TAK1; and protein linase B(PKB, also called Akt). Moreover, the downstream pathway of TRIP-1 isthe stress-activated MAP kinase pathway, required for cell cycle stressresponse Humphrey T, Enoch T Genetics 148:1731-42, (1998); MacDonald RG, Science 239:1134-7, (1988)).

Disclosed herein, ras and raf, the upstream regulators of MEKK affectedthe TRAP-induced apoptosis. Overexpression dominant negative (DN)-Rasand DN-Raf in the ROS 17/2.8 cells eliminated the TRAP apoptosis effect.Over-expression of Ras signaling can decrease the protein amount ofintegrin and increase the expression level of Fas in thetransfected-osteoblast (MacDonald R G, Science 239:1134-7, (1988)).

Disclosed herein osteoclast (TRAP staining +) and aged osteoblast cells(senescence-associated β-galactosidase staining positive) are colocalizeto the same area. Most of the SA-βgal positive cells are located in thegrowth plate area of long bone. This epiphysial growth plate containsmost of the trabaculae bone and most of the bone remodeling happens inthis area.

C. Compositions 1. Compositions That Bind Tartrate Resistant AcidPhosphatase TRAP

Disclosed are compositions that bind TRAP. Unless otherwise indicatedthe binding of compositions to TRAP can occur in any way that promotesan interaction between the composition and TRAP that is greater than anon-specific interaction. A non-specific interaction is defined as aninteraction between TRAP and bovine serum albumin. Typically thecomposition has a K_(d) for TRAP less than or equal to 10⁻⁵ M. In otherembodiments the K_(d) for TRAP is less than or equal to 10⁻⁶ M or theK_(d) for TRAP is less than or equal to 10⁻⁷ M or the K_(d) for TRAP isless than or equal to 10⁻⁸ M or the K_(d) for TRAP is less than or equalto 10⁻⁹ M or the K_(d) for TRAP is less than or equal to 10⁻¹⁰ M or theK_(d) for TRAP is less than or equal to 10⁻¹¹ M or the K_(d) for TRAP isless than or equal to 10⁻¹² M. An isolated nucleic acid moleculecomprising a sequence encoding a peptide that binds tartrate resistantacid phosphatase is disclosed.

The composition can be determined to have an interaction with TRAPgreater than a non-specific interaction by performing the followingassays. TRAP binding affinity can be measured by use of a Scatchardanalysis with TRAP as the immobilized substrate and the target proteinas the ligand. The target protein can be expressed in phage and thephage can be detected with anti-phage antibodies. Calculation of theVmax and Kd for binding would define the interaction. TRAP binding canalso be measured with Western and Far-Western technology. Proteinsseparated on a gel and transferred to a membrane can be probed withTRAP. Detection of TRAP binding to specific proteain bands can beaccomplished with antibodies to TRAP or phage expressing proteins withaffinity for TRAP.

Disclosed are isolated nucleic acid molecules comprising a sequenceencoding a means for binding tartrate resistant acid phosphatase and aregion controlling the expression of the means for binding tartrateresistant acid phosphatase.

Also disclosed are isolated nucleic acid molecules comprising a sequenceencoding an integration site and a means for binding tartrate resistantacid phosphatase.

Also disclosed are isolated nucleic acid molecules comprising a sequenceencoding a replication site and a means for binding tartrate resistantacid phosphatase.

The means for binding TRAP can be any means capable of binding TRAP asdiscussed herein. For example, the peptides disclosed in SEQ ID NOs:19-36, in particular SEQ ID NO:23 are means for binding TRAP.

Also disclosed are means for binding TRAP that do not comprise thepeptides having the sequence set forth in SEQ ID NOs: 38 and 40.

2. Compositions that Bind Osteoblasts, Osteoclasts, and/or OsteoclastLacuna

Disclosed are compositions that bind osteoblasts, osteoclasts, and/orosteoclast lacuna. Unless otherwise indicated the binding ofcompositions to osteoblasts, osteoclasts, and/or osteoclast lacuna canoccur in any way that promotes an interaction between the compositionand osteoblasts, osteoclasts, and/or osteoclast lacuna that is greaterthan a non-specific interaction. A non-specific interaction is definedas an interaction between osteoblasts, osteoclasts, and/or osteoclastlacuna and bovine serum albumin. Typically the composition has a K_(d)for osteoblasts, osteoclasts, and/or osteoclast lacuna less than orequal to 10⁻⁵ M. In other embodiments the K_(d) for osteoblasts,osteoclasts, and/or osteoclast lacuna is less than or equal to 10⁻⁶ M orthe K_(d) for osteoblasts, osteoclasts, and/or osteoclast lacuna is lessthan or equal to 10⁻⁷ M or the K_(d) for osteoblasts, osteoclasts,and/or osteoclast lacuna is less than or equal to 10⁻⁸ M or the K_(d)for osteoblasts, osteoclasts, and/or osteoclast lacuna is less than orequal to 10⁻⁹ M or the K_(d) for osteoblasts, osteoclasts, and/orosteoclast lacuna is less than or equal to 10⁻¹⁰ M or the K_(d) forosteoblasts, osteoclasts, and/or osteoclast lacuna is less than or equalto 10⁻¹¹ M or the K_(d) for osteoblasts, osteoclasts, and/or osteoclastlacuna is less than or equal to 10⁻¹² M. An isolated nucleic acidmolecule comprising a sequence encoding a peptide that binds tartrateresistant acid phosphatase.

The composition can be determined to have an interaction withosteoblasts, osteoclasts, and/or osteoclast lacuna greater than anon-specific interaction by performing the following assays.Osteoblasts, osteoclasts, and/or osteoclast lacuna binding affinity forthe composition can be measured by use of a Scatchard analysis withosteoblasts, osteoclasts, and/or osteoclast lacuna as the immobilizedsubstrate and the target protein as the ligand. The target protein canbe expressed in phage and the phage can be detected with anti-phageantibodies. Calculation of the Vmax and Kd for binding would define theinteraction. Osteoblasts, osteoclasts, and/or osteoclast lacuna bindingcan also be measured with Western and Far-Western technology. Proteinsseparated on a gel and transferred to a membrane can be probed withosteoblasts, osteoclasts, and/or osteoclast lacuna. Detection ofosteoblasts, osteoclasts, and/or osteoclast lacuna binding to specificproteain bands can be accomplished with antibodies to osteoblasts,osteoclasts, and/or osteoclast lacuna or phage expressing proteins withaffinity for osteoblasts, osteoclasts, and/or osteoclast lacuna.

Disclosed are isolated nucleic acid molecules comprising a sequenceencoding a means for binding osteoblasts, osteoclasts, and/or osteoclastlacuna and a region controlling the expression of the means for bindingtartrate resistant acid phosphatase.

Also disclosed are isolated nucleic acid molecules comprising a sequenceencoding an integration site and a means for binding osteoblasts,osteoclasts, and/or osteoclast lacuna.

Also disclosed are isolated nucleic acid molecules comprising a sequenceencoding a replication site and a means for binding osteoblasts,osteoclasts, and/or osteoclast lacunae.

The means for binding osteoblasts, osteoclasts, and/or osteoclast lacunacan be any means capable of binding osteoblasts, osteoclasts, and/orosteoclast lacuna as discussed herein. For example, the peptidesdisclosed in SEQ ID NOs: 19-36, in particular SEQ ID NO:23 are means forbinding osteoblasts, osteoclasts, and/or osteoclast lacuna.

Also disclosed are means for binding osteoblasts, osteoclasts, and/orosteoclast lacuna that do not comprise the peptides having the sequenceset forth in SEQ ID NOs: 38 and 40.

3. Implants

The disclosed peptides and proteins can be attached to implants, such asbone and dental implants. A bone or dental implant is an object that iscapable of being attached or fixed to bone. There are many differenttypes of bone implants for many different situations and conditions. Inaddition there are many different ways to attach the implants to thebone or enhance the attachement of the implant to the bone. Likewisethere are many different materials which can serve as implants, and manydifferent types of devices can be attached to omplants. It is understoodthat any implant can be modified as discussed herein by the addition ofthe TRAP and TRAP variants, or the GPC4 or GPC4 variants or TRIP or TRIPvariants or peptide or peptide variants as discussed herein. Thedisclosed compositions and methods help in implant attachment and thestimulation of bone growth in and around the implant because thedisclosed compositions and methods in certain embodiments promoterecruitment of osteoblasts and differentiation of osteoblasts. Examplesof different bone implants and various materials and methods and devicesrelated to implants can be found in the following U.S. patents, all ofwhich are incorporated by reference for material related to implants:U.S. Pat. No. 6,447,545, Self-aligning bone implant;” U.S. Pat. No.6,440,444, “Load bearing osteoimplant and method of repairing bone usingthe same;” U.S. Pat. No. 6,425,920, “Spinal fusion implant;” U.S. Pat.No. 6,413,089, “Immediate post-extraction implant;” U.S. Pat. No.6,409,730, “Humeral spiral blade;” U.S. Pat. No. 6,406,296, “Implantwith enlarged proximal segment;” U.S. Pat. No. 6,398,786,“Strain-inducing conical screw for stimulating bone transplant growth;”U.S. Pat. No. 6,379,153, “Dental implant having a dual-textured exteriorsurface;” U.S. Pat. No. 6,370,418, “Device and method for measuring theposition of a bone implant;” U.S. Pat. No. 6,364,663, “Tooth implant andmethod to make it;” U.S. Pat. No. 6,350,283, “Bone hemi-lumbar interbodyspinal implant having an asymmetrical leading end and method ofinstallation thereof;” U.S. Pat. No. 6,350,126, “Bone implant;” U.S.Pat. No. 6,343,930, “Ceramic dental abutments with a metallic core;”U.S. Pat. No. 6,312,468, “Silicon-substituted apatites and process forthe preparation thereof, “;” U.S. Pat. No. 6,309,220, “Bone distentionand condensation dental implant distractor apparatus and method;” U.S.Pat. No. 6,302,913, “Biomaterial and bone implant for bone repair andreplacement;” U.S. Pat. No. 6,287,115, “Dental implant and tool andmethod for effecting a dental restoration using the same;” U.S. Pat. No.6,283,997, “Controlled architecture ceramic composites bystereolithography;” U.S. Pat. No. 6,280,478, “Artefact suitable for useas a bone implant;” U.S. Pat. No. 6,267,785, “Apparatus for positioninga prosthetic element to achieve a desired orientation for cementation;”U.S. Pat. No. 6,264,656, “Threaded spinal implant;” U.S. Pat. No.6,261,288, “Implant stabilization and locking system;” U.S. Pat. No.6,241,769, “Implant for spinal fusion;” U.S. Pat. No. 6,238,435,“Assembly tool for prosthetic implant;” U.S. Pat. No. 6,227,858, “Boneanchoring element;” U.S. Pat. No. 6,221,111, “Bioactive surface layerfor bone implants;” U.S. Pat. No. 6,217,617, “Bone implant and method ofsecuring;” U.S. Pat. No. 6,213,775, “Method of fastening an implant to abone and an implant therefor;” U.S. Pat. No. 6,206,924,“Three-dimensional geometric bio-compatible porous engineered structurefor use as a bone mass replacement or fusion augmentation device;” U.S.Pat. No. 6,193,762, “Surface for use on an implantable device;” U.S.Pat. No. 6,193,719, “Threaded clamping plug for interconnecting twoimplants of a spinal osteosynthesis instrumentation or other implants;”U.S. Pat. No. 6,187,009, “Osteosynthesis implant;” U.S. Pat. No.6,168,436, “Universal dental implant abutment system;” U.S. Pat. No.6,168,435, “Ceramic dental abutments with a metallic core;” U.S. Pat.No. 6,162,258, “Lyophilized monolithic bone implant and method fortreating bone;” U.S. Pat. No. 6,149,432, “Buttress thread dentalimplant;” U.S. Pat. No. 6,146,384, “Orthopedic fixation device andmethod of implantation;” U.S. Pat. No. 6,136,038, “Bone connectiveprosthesis and method of forming same;” U.S. Pat. No. 6,096,080,“Apparatus for spinal fusion using implanted devices;” U.S. Pat. No.6,090,998, “Segmentally demineralized bone implant;” U.S. Pat. No.6,074,394, “Targeting device for an implant;” U.S. Pat. No. 6,059,832,“Prosthetic wrist implants, instruments, and related methods ofimplantation;” U.S. Pat. No. 6,058,590, “Apparatus and methods forembedding a biocompatible material in a polymer bone implant;” U.S. Pat.No. 6,056,750, “Fixing element for osteosynthesis;” U.S. Pat. No.6,048,344, “Threaded washer and bone screw apparatus;” U.S. Pat. No.6,046,164, “Therapeutic agent for diseases of periodontal tissue;” U.S.Pat. No. 6,032,677, “Method and apparatus for stimulating the healing ofmedical implants;” U.S. Pat. No. 6,025,538,” Compound bone structurefabricated from allograft tissue;”, “U.S. Pat. No. 6,004,327,“Ratcheting compression device;” U.S. Pat. No. 5,976,149, “Method andapparatus for aligning a prosthetic element;” U.S. Pat. No. 5,964,766,“Buttress thread implant;” U.S. Pat. No. 5,961,328, “Dental implant;”U.S. Pat. No. 5,951,564, “Orthopaedic positioning apparatus;” U.S. Pat.No. 5,944,721, “Method for repairing fractured bone;” U.S. Pat. No.5,938,443, “Impression coping for use in an open tray and closed trayimpression methodology;” U.S. Pat. No. 5,921,774, “Supporting body foruse in orthodontic appliance and method;” U.S. Pat. No. 5,895,425, “Boneimplant;” U.S. Pat. No. 5,885,287, “Self-tapping interbody boneimplant;” U.S. Pat. No. 5,881,443, “Apparatus and methods for embeddinga biocompatible material in a polymer bone implant;” U.S. Pat. No.5,876,453, “Implant surface preparation;” U.S. Pat. No. 5,842,865,“Self-tapping implant with multiple concave tapping channels;” U.S. Pat.No. 5,836,768, “Fastening device for fixing orthodontic apparatuses on adental implant;” U.S. Pat. No. 5,823,777, “Dental implants to optimizecellular response;” U.S. Pat. No. 5,800,550, “Interbody fusion cage;”U.S. Pat. No. 5,788,976, “Method for effecting bone repair;” U.S. Pat.No. 5,769,854, “Instrument system for preparing a distal femur for aposteriorly stabilized femoral component of a knee prosthesis;” U.S.Pat. No. 5,755,799, “Joint implant with self-engaging attachmentsurface;” U.S. Pat. No. 5,741,256, “Helical osteosynthetic implant;”U.S. Pat. No. 5,739,176, “Biodegradable in-situ forming implants andmethods of producing the same;” U.S. Pat. No. 5,738,521, “Method foraccelerating osseointegration of metal bone implants using electricalstimulation;” U.S. Pat. No. 5,709,547, “Dental implant for anchorage incortical bone;” U.S. Pat. No. 5,702,695, “Osseointegration promotingimplant composition, implant assembly and method therefor;” U.S. Pat.No. 5,702,475, “Modular bone implant with pan and pins;” U.S. Pat. No.5,702,470, “Prosthetic wrist implant and related method ofimplantation;” U.S. Pat. No. 5,697,779, “Temporary implant for use as ananchor in the mouth;” U.S. Pat. No. 5,693,099, “Endoprosthesis;” U.S.Pat. No. 5,693,098, “Fibrin D-domain multimer prostheses and methods fortheir production;” U.S. Pat. No. 5,691,305, “Bone implant compositioncomprising a porous matrix, bone growth promoter proteins, andphosphotyrosyl protein phosphatase inhibitor;” U.S. Pat. No. 5,674,288,“Implant with transponder marker;” U.S. Pat. No. 5,665,089, “Bonefixation system;” U.S. Pat. No. 5,628,630, “Design process for skeletalimplants to optimize cellular response;” U.S. Pat. No. 5,626,579, “Bonetransport and lengthening system;” U.S. Pat. No. 5,624,462, “Boneimplant and method of securing;” U.S. Pat. No. 5,609,636, “Spinalimplant;” U.S. Pat. No. 5,609,631, “Fibrin D-domain multimer coatedprostheses and methods for their production;” U.S. Pat. No. 5,601,558,“Soft tissue anchors and systems for implantation;” U.S. Pat. No.5,580,246, “Dental implants and methods for extending service life;”U.S. Pat. No. 5,573,401, “Biocompatible, low modulus dental devices;”U.S. Pat. No. 5,571,198, “Acetabular shell with selectively availablebone screw holds;” U.S. Pat. No. 5,571,185, “Process for the productionof a bone implant and a bone implant produced thereby;” U.S. Pat. No.5,564,925, “Implant for an artificial tooth;” U.S. Pat. No. 5,558,517,“Polymeric prosthesis having a phosphonylated surface;” U.S. Pat. No.5,555,884, “Measuring method by using resonance of a resonance medium;”U.S. Pat. No. 5,545,226, “Implants for cranioplasty;” U.S. Pat. No.5,542,847, “Method, apparatus and device for dental prosthesisimplantation;” U.S. Pat. No. 5,523,348, “Method of preparingcollagen-polymer conjugates;” U.S. Pat. No. 5,514,137, “Fixation oforthopedic devices;” U.S. Pat. No. 5,507,829, “Set of fixation stemshaving similar stiffness;” U.S. Pat. No. 5,507,815, “Random surfaceprotrusions on an implantable device;” U.S. Pat. No. 5,503,558,“Osseointegration promoting implant composition, implant assembly andmethod therefor;” U.S. Pat. No. 5,449,291, “Dental implant assemblyhaving tactile feedback;” U.S. Pat. No. 5,397,358, “Bone implant;” U.S.Pat. No. 5,397,235, “Method for installation of dental implant;” U.S.Pat. No. 5,395,374, “Orthopedic cabling method and apparatus;” U.S. Pat.No. 5,387,243, “Method for converting a cementable implant to a pressfit implant;” U.S. Pat. No. 5,370,693, “Orthopedic implant augmentationand stabilization device;” U.S. Pat. No. 5,336,465, “Method of makingbone-implants;” U.S. Pat. No. 5,306,306, “Method for periprosthetic bonemineral density measurement;” U.S. Pat. No. 5,300,120, “Implant withelectrical transponder marker;” U.S. Pat. No. 5,290,291, “Method forimplant removal;” U.S. Pat. No. 5,282,746, “Method of installing adental prosthesis;” U.S. Pat. No. 5,258,098, “Method of production of asurface adapted to promote adhesion;” U.S. Pat. No. 5,236,459, “Boneimplant and method of making same;” U.S. Pat. No. 5,236,359, “Tappingtool and method for implant dentistry;” U.S. Pat. No. 5,221,204, “Dentalimplant product and method of making;” U.S. Pat. No. 5,201,735,“Apparatus and method for treating a fracture;” U.S. Pat. No. 5,190,546,“Medical devices incorporating SIM alloy elements;” U.S. Pat. No.5,190,544, “Modular femoral fixation system;” U.S. Pat. No. 5,181,926,“Bone implant having relatively slidable members;” U.S. Pat. No.5,171,327, “Bone implant;” U.S. Pat. No. 5,147,408, “Prosthetic deviceand method of implantation;” U.S. Pat. No. 5,108,453, “Bone implant;”U.S. Pat. No. 5,084,050, “Implant for bone reinforcement and foranchoring bone screws, implants and implant parts;” U.S. Pat. No.5,066,296, “Apparatus for treating a fracture;” U.S. Pat. No. 5,024,239,“Method and apparatus for determining osseous implant fixationintegrity;” U.S. Pat. No. 5,024,232, “Novel radiopaque heavy metalpolymer complexes, compositions of matter and articles preparedtherefrom;” U.S. Pat. No. 5,013,314, “Instrumentation and method forinserting flexible implants into fractured bones;” U.S. Pat. No.5,006,070, “Dental implant with y-shaped body;” U.S. Pat. No. 5,002,580,“Prosthetic device and method of implantation;” U.S. Pat. No. 5,002,575,“Bone implant prosthesis;” U.S. Pat. No. 4,997,383, “Dental implant;”U.S. Pat. No. 4,990,163, “Method of depositing calcium phosphatecermamics for bone tissue calcification enhancement;” U.S. Pat. No.4,990,161, “Implant with resorbable stem;” U.S. Pat. No. 4,978,355,“Plastic bone implant having a reinforced contact surface;” U.S. Pat.No. 4,976,739, “Implant system;” U.S. Pat. No. 4,976,738, “Porous metaloverlay for an implant surface;” U.S. Pat. No. 4,969,908, “Lunateimplant and method of stabilizing same;” U.S. Pat. No. 4,969,907, “Metalbone implant;” U.S. Pat. No. 4,969,904, “Bone implant;” U.S. Pat. No.4,955,911, “Bone implant;” U.S. Pat. No. 4,936,855, “Stepped-lock ringsystem for implantable joint prostheses;” U.S. Pat. No. 4,936,851,“Analytic bone implant;” U.S. Pat. No. 4,919,666, “Implant havingrecesses for therapeutically effective substances;” U.S. Pat. No.4,904,264, “Artifical joint system;” U.S. Pat. No. 4,904,187, “Dentalimplant;” U.S. Pat. No. 4,883,492, “Metal bone implant;” U.S. Pat. No.4,877,020, “Apparatus for bone graft;” U.S. Pat. No. 4,865,602, “Gammairradiation of collagen/mineral mixtures;” U.S. Pat. No. 4,854,873,“Oral implant;” U.S. Pat. No. 4,851,008, “Bone implant prosthesis withsubstantially stress-free outer surface;” U.S. Pat. No. 4,842,606, “Boneimplant;” U.S. Pat. No. 4,834,757, “Prosthetic implant;” U.S. Pat. No.4,828,563, “Implant;” U.S. Pat. No. 4,801,299, “Body implants ofextracellular matrix and means and methods of making and using suchimplants;” U.S. Pat. No. 4,800,639, “Method of making a metal boneimplant;” U.S. Pat. No. 4,793,335, “Bone implant for fixing artificialtendons or ligaments with application and extraction means;” U.S. Pat.No. 4,790,849, “Malar implant and method of inserting the prothesis;”U.S. Pat. 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No. 4,012,796,“Interpositioning collar for prosthetic bone insert;” U.S. Pat. No.4,000,525, “Ceramic prosthetic implant suitable for a knee jointplateau;” U.S. Pat. No. 3,981,079, “Dental implant and method ofmounting the same in the jaw bone;” and U.S. Pat. No. 3,973,277,“Attaching fibrous connective tissue to bone.”

It is understood that in certain embodiments, the proteins or peptidesattached to the implant can be cleavable from the implant. In theseembodiments the peptide or protein is attached to the implant such thatthe peptide or protein can be removed from the implant over time or incertain conditions. For example, this type of embodiment can furtherenhance diffusion of the molecules for the purpose of inducingdifferentiation of osteoblasts near the implant because it allows forthe importation of the TRAP or TRAP fragment or TRAP like molecule intothe cell to promote interaction with TRIP. There are many systems andreagents for attaching proteins or peptides to surfaces in anon-permanent manner.

For example, U.S. Pat. No. 6,416,758, which is herein incorporated byreference for material related to linkers for peptides discussesbiochemical cross linkers and peptidase cross linkers. U.S. Pat. No.6,416,758 provides different heterofunctional biochemical cross linkerswhich can be used to link peptides to other peptide or surfaces whichare able to react with one of the reactive groups on the cross linker.For example, various hetero-bi-functional cross linkers discussed inU.S. Pat. No. 6,416,758 are SMPT, SPDP, LC-SPDP, Sulfo-LC-SPDP, SMCC,Sulfo-SMCC, MBS, Sulfo-MBS, SIAB, Sulfo-SIAB, SMPB, Sulfo-SMPB,EDC/Sulfo-NHS, and ABH. The key aspect of hetero-bi-functional linkersis that there is typically one group that reacts with a primary amine(e.g., N-hydroxy succinimide), for example, the primary amine present onmost proteins and petides, and another group that reacts with a thiolgroup (e.g., pyridyl disulfide, maleimides, halogens, etc.), which couldbe for example, present on the implant. For example, primary orsecondary amine groups, hydrazide or hydrazine groups, carboxyl alcohol,phosphate, or alkylating groups may be used for binding orcross-linking.

It is understood that the linking groups may have a spacer arm, orlinker, between them, which may be any length appropriate for thesituation. These various cross linkers and others have a variety ofstabilities in vivo, in the presence of blood, for example, and linkerscan be utilized which have more or less stability in vivo, to achieve alesser or greater release respectively. These types of linkers aretermed biologically releaseable linkers. For example, linkers can beutilized which are acid cleavable. A biologically-releasable linkerincludes all linkages that are releasable, cleavable or hydrolyzableonly or preferentially under certain conditions. This includes disulfideand trisulfide bonds and acid-labile bonds, as described in U.S. Pat.Nos. 5,474,765 and 5,762,918, each specifically incorporated herein byreference at least for material related to linkers. Biologicallyreleasable linkers can also have enzyme-sensitive bonds, includingpeptide bonds, esters, amides, phosphodiesters and glycosides, whichallow for the cleavage by enzymes or environmental conditions. Forexample, there are numerous proteases that exist, particularly at thesite bone resorption and formation, and these proteases are typicallyactivated in an acidic environment. There are also many proteases t tare circulating, such as proteases involved in the blood coagulationsystem. For example, peptide linkers that include a cleavage site forurokinase, pro-urokinase, plasmin, plasminogen, TGF.beta.,staphylokinase, Thrombin, Factor IXa, Factor Xa or a metalloproteinase,such as an interstitial collagenase, a gelatinase or a stromelysin,exist and are discussed in U.S. Pat. Nos. 6,004,555 and 5,877,289 whichare herein incorporated by reference at least for material related tolinkers and biologically releasable bonds. These patents discuss the useof linkers typically in the context of tumor cell envronments, but canbe adapted for use with the disclosed implants. An exemplary list ofcleavable linker sequences set forth in U.S. Pat. No. 6,416,758 is:Plasmin cleavable sequences, such as Pro-urokinase (PRFKIIGG, SEQ IDNO:51, PRFRIIGG, SEQ ID NO:52); TGFβ (SSRHRRALD, SEQ ID NO:53);Plasminogen (RKSSIIIRMRDVVL, SEQ ID NO:54); Staphylokinase(SSSFDKGKYKKGDDA, SEQ ID NO:55, SSSFDKGKYKRGDDA, SEQ ID NO:56); FactorXa cleavable sequences (IEGR, SEQ ID NO:57, IDGR, SEQ ID NO:58, GGSIDGR,SEQ ID NO:59); MMP cleavable sequences, such as Gelatinase A (PLGLWA,SEQ ID NO:60); Collagenase cleavable sequences such as Calf skincollagen (α1(I) chain) (GPQGIAGQ, SEQ ID NO:61), Calf skin collagen(α2(I) chain) (GPQGLLGA, SEQ ID NO:62), Bovine cartilage collagen(α1(II) chain) (GIAQQ, SEQ ID NO:63); Human liver collagen (α1(III)chain) (QPLGTIAGI, SEQ ID NO:64), Human α₂ M (GPEGLRVG, SEQ ID NO:65),Human PZP (YGAGLGVV, SEQ ID NO:66, AGLGVVER, SEQ ID NO:67, AGLGISST, SEQID NO:68), Rat α₁ M (EPQALAMS, SEQ ID NO:69, QALAMSAI, SEQ ID NO:70),Rat α₂ M (AAYHLVSQ, SEQ ID NO:71, MDAFLESS, SEQ ID NO:72), Rat α₁ I₃(2J) (ESLPVVAV, SEQ ID NO:73), Rat α₁ I₃ (27J) (SAPAVESE, SEQ ID NO:74),Human fibroblast collagenase (autolytic cleavages) (DVAQFVLT, SEQ IDNO:75, VAQFVLTE, SEQ ID NO:76, AQFVLTEG, SEQ ID NO:77, PVQPIGPQ, SEQ IDNO:78). These are examples of cleavable peptide linkers and they can beadapted for use with the disclosed implants and compositions.

It is understood that any biologically releasable linker can be usedwith the implants disclosed herein, as long as the linkers allow for therelease of the peptide or protein such that it can interact with TRAP,TRIP, or GPC4.

4. Nucleic Acids

Nucleic acid molecules that encode peptides that bind TRAP andosteoclast lacuna are disclosed. Nucleic acid molecules that encodepeptides that can inhibit osteoblasts from interacting with osteoclastlacuna are disclosed. Also disclosed are nucleic acid molecules thatencode proteins expressed in osteoblasts, such as TRIP and GPC4, whichinteract with TRAP.

Disclosed are isolated nucleic acid molecules comprising a sequence setforth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6,SEQ ID NO:7,SEQ ID NO:8,SEQ ID NO:9,SEQ ID NO:10,SEQID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:37, SEQ ID NO:39, or SEQID NO:41.

Disclosed are isolated nucleic acid molecules comprising a complement toa sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:37,SEQ ID NO:39, or SEQ ID NO:41.

Also disclosed are isolated nucleic acid molecules comprising a sequencehaving at least 80% identity to a sequence set forth in SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, or SEQ ID NO:37, SEQ ID NO:39, or SEQ ID NO:41.

Disclosed are isolated nucleic acid molecules comprising a sequencehaving at least 80% identity to a complement to a sequence set forth inSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17 SEQ ID NO:18, or SEQ ID NO:37, SEQ ID NO:39, or SEQ IDNO:41.

Disclosed are isolated nucleic acid molecules comprising a sequence thathybridizes under stringent hybridization conditions to a sequence setforth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:37, SEQ ID NO:39,or SEQ ID NO:41.

Also disclosed are isolated nucleic acid molecules comprising a sequencethat hybridizes under stringent hybridization conditions to a complementof a sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:37,SEQ ID NO:39, or SEQ ID NO:41.

Disclosed are isolated nucleic acid molecules comprising a sequenceencoding a peptide set forth in SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:38, SEQ ID NO:40, or SEQ ID NO:42.

Disclosed are isolated nucleic acid molecules comprising a sequenceencoding a peptide having at least 80% identity to a peptide set forthin SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:40, orSEQ ID NO:42.

Also disclosed are isolated nucleic acid molecules comprising a sequenceencoding a peptide having at least 80% identity to a peptide set forthin SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:40, orSEQ ID NO:42, wherein any change from SEQ ID Nos:19-36, SEQ ID NO:35,SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:42. is a conservative change.

Also disclosed are isolated nucleic acid molecules that encode apeptide, wherein the encoded peptide binds an osteoclast cell, isolatednucleic acid molecules that encode a peptide, wherein the encodedpeptide binds an osteoclast lacuna, isolated nucleic acid molecules thatencode a peptide, wherein the encoded peptide binds a lysosomal proteinexpressed in osteoclasts, and isolated nucleic acid molecules, whereinthe encoded peptide binds the lysosomal protein, tartrate resistant acidphosphatase. Other known lysosomal proteins are for example, the familyof cathepsin enzymes, galactosidase and glucosidase enzymes, lysozyme,tartrate sensitive acid phosphatase (TSAP).

Disclosed are isolated nucleic acid molecules, wherein the encodedpeptide binds with a K_(d) less than or equal to 10⁻⁵, less than orequal to 10⁻⁶, less than or equal to 10⁻⁷, less than or equal to 10⁻⁸,less than or equal to 10⁻⁹, less than or equal to 10⁻¹⁰, less than orequal to 10⁻¹¹, or less than or equal to 10⁻¹².

Disclosed are primers comprising a sequence set forth in SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:37, SEQ ID NO:39, or SEQ ID NO:41 or aportion thereof.

Disclosed are primers comprising a sequence which is a complement to asequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:37,SEQ ID NO:39, or SEQ ID NO:41 or a portion thereof.

Disclosed are primers comprising a sequence set forth in SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:37, SEQ ID NO:39, or SEQ ID NO:41.

Disclosed are primers comprising a sequence which is a complement to asequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:37,SEQ ID NO:39, or SEQ ID NO:41.

Disclosed are primers and probes that comprise a potion of the sequenceset forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:37, SEQ ID NO:39, orSEQ ID NO:41.

Disclosed are vectors comprising a nucleic acid set forth in any one ofclaims SEQ ID NO:1, SEQ ID NO:2, SEQ D NO:3, SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:37, SEQ ID NO:39, or SEQ IDNO:41.

Also disclosed are vectors comprising a nucleic acid having at least a80%, 85%, 90, or 95% identity to or hybridize under stringent conditionsto a sequence set forth in any one of claims SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ D NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:37, SEQ ID NO:39, or SEQ ID NO:41.

Also disclosed are cells comprising any of the non-naturally occurringdisclosed nucleic acids. Non-naturally occurring means occurring in acell in a way that has been affected by a recombinant molecular biologytechnique or delivery of the composition to the cell.

Also disclosed are cells, wherein the cell is selected from the groupconsisting of a fibroblast cell, a cartilage cell, a bone cell, bonemarrow cell, a stem cell, and an adipocyte cell, osteoblast cell.

Also disclosed are cells, wherein the exogenous nucleic acid in the cellis under the control of a cell specific promoter. Also disclosed arecells, wherein the cell specific promoter is a bone cell specificpromoter such as, type I collagen, alkaline phosphatase, osteonectin,osteocalcin, and the cbfa1 promoter.

Also disclosed are implants for promoting bone growth, wherein theimplant comprises the cells disclosed herein or the peptides andproteins disclosed herein.

Also disclosed are animals comprising any of the non-naturally occurringnucleic acids disclosed.

Also disclosed are microarrays comprising any of the nucleic acidsdisclosed herein.

Disclosed are pharmaceutical compositions comprising any of the nucleicacids disclosed herein along with pharmaceutically acceptable carrier.

Also disclosed are isolated nucleic acid molecules, wherein the identityis at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% to any of the disclosed nucleic acidsequences wherein the disclosed nucleic acid sequence is not SEQ IDNOs:37, 39, and 41.

a) Sequence Similarities

Disclosed are nucleic acids and peptides that are involved ininteractions and modulating interactions that can occur betweenosteoblasts and osteoclasts/osteoclast lacuna, and peptides that areexpressed by osteoblasts and osteoclasts. It is understood that thedisclosed nucleic acids and peptides can be defined by the homology oridentity that they have to particular sequences or variants of thedisclosed compositions.

It is understood that as discussed herein the use of the terms homologyand identity mean the same thing as similarity. Thus, for example, ifthe use of the word homology is used between two non-natural sequencesit is understood that this is not necessarily indicating an evolutionaryrelationship between these two sequences, but rather is looking at thesimilarity or relatedness between their nucleic acid sequences. Many ofthe methods for determining homology between two evolutionarily relatedmolecules are routinely applied to any two or more nucleic acids orproteins for the purpose of measuring sequence similarity regardless ofwhether they are evolutionarily related or not.

In general, it is understood that one way to define any known variantsand derivatives or those that might arise, of the disclosed genes andproteins herein, is through defining the variants and derivatives interms of homology to specific known sequences. This identity ofparticular sequences disclosed herein is also discussed elsewhereherein. In general, variants of genes and proteins herein disclosedtypically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, or 99 percent homology to the stated sequence or the nativesequence. Those of skill in the art readily understand how to determinethe homology of two proteins or nucleic acids, such as genes. Forexample, the homology can be calculated after aligning the two sequencesso that the homology is at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment. It isunderstood that any of the methods typically can be used and that incertain instances the results of these various methods may differ, butthe skilled artisan understands if identity is found with at least oneof these methods, the sequences would be said to have the statedidentity, and be disclosed herein.

For example, as used herein, a sequence recited as having a particularpercent homology to another sequence refers to sequences that have therecited homology as calculated by any one or more of the calculationmethods described above. For example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingthe Zuker calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by any of theother calculation methods. As another example, a first sequence has 80percent homology, as defined herein, to a second sequence if the firstsequence is calculated to have 80 percent homology to the secondsequence using both the Zuker calculation method and the Pearson andLipman calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by the Smith andWaterman calculation method, the Needleman and Wunsch calculationmethod, the Jaeger calculation methods, or any of the other calculationmethods. As yet another example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingeach of calculation methods (although, in practice, the differentcalculation methods will often result in different calculated homologypercentages).

b) Hybridization/Selective Hybridization

Disclosed are nucleic acids and it is understood that nucleic acids havecertain properties, among them the ability to interact in a sequencespecific manner, termed hybridization. The level or amount ofhybridization that occurs between nucleic acids is understood to be oneway to define the relationship between those two nucleic acids.

The term hybridization typically means a sequence driven interactionbetween at least two nucleic acid molecules, such as a primer or a probeand a gene. Sequence driven interaction means an interaction thatoccurs, for example, between two nucleotides or nucleotide analogs ornucleotide derivatives or nucleotide substitutions, in a nucleotidespecific manner. For example, G interacting with C or A interacting withT are sequence driven interactions. Typically sequence driveninteractions occur on the Watson-Crick face or Hoogsteen face of thenucleotide. The hybridization of two nucleic acids is affected by anumber of conditions and parameters known to those of skill in the art.For example, the salt concentrations, pH, and temperature of thereaction all affect whether two nucleic acid molecules will hybridize.

Parameters for selective hybridization between two nucleic acidmolecules are well known to those of skill in the art. For example, insome embodiments selective hybridization conditions can be defined asstringent hybridization conditions. For example, stringency ofhybridization is controlled by both temperature and salt concentrationof either or both of the hybridization and washing steps. For example,the conditions of hybridization to achieve selective hybridization mayinvolve hybridization in high ionic strength solution (6×SSC or 6×SSPE)at a temperature that is about 12-25° C. below the Tm (the meltingtemperature at which half of the molecules dissociate from theirhybridization partners) followed by washing at a combination oftemperature and salt concentration chosen so that the washingtemperature is about 5° C. to 20° C. below the Tm. The temperature andsalt conditions are readily determined empirically in preliminaryexperiments in which samples of reference DNA immobilized on filters arehybridized to a labeled nucleic acid of interest and then washed underconditions of different stringencies. Hybridization temperatures aretypically higher for DNA-RNA and RNA-RNA hybridizations. The conditionscan be used as described above to achieve stringency, or as is known inthe art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rdEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2001;Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which are hereinincorporated by reference for material at least related to hybridizationof nucleic acids). A preferable stringent hybridization condition for aDNA:DNA hybridization can be at about 68° C. (in aqueous solution) in6×SSC or 6×SSPE followed by washing at 68° C. Stringency ofhybridization and washing, if desired, can be reduced accordingly as thedegree of complementarity desired is decreased, and further, dependingupon the G-C or A-T richness of any area wherein variability is searchedfor. Likewise, stringency of hybridization and washing, if desired, canbe increased accordingly as homology desired is increased, and further,depending upon the G-C or A-T richness of any area wherein high homologyis desired, all as known in the art.

Another way to define selective hybridization is by looking at theamount (percentage) of one of the nucleic acids bound to the othernucleic acid. For example, in some embodiments selective hybridizationconditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid isbound to the non-limiting nucleic acid. Typically, the non-limitingprimer is in for example, 10 or 100 or 1000 fold excess. This type ofassay can be performed under conditions where both the limiting andnon-limiting primer are for example, 10 fold or 100 fold or 1000 foldbelow their K_(d), or where only one of the nucleic acid molecules is 10fold or 100 fold or 1000 fold or where one or both nucleic acidmolecules are above their K_(d).

Another way to define selective hybridization is by looking at thepercentage of primer that gets enzymatically manipulated underconditions where hybridization is required to promote the desiredenzymatic manipulation. For example, in some embodiments selectivehybridization conditions would be when at least about, 60, 65, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer isenzymatically manipulated under conditions which promote the enzymaticmanipulation, for example if the enzymatic manipulation is DNAextension, then selective hybridization conditions would be when atleast about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100percent of the primer molecules are extended. Preferred conditions alsoinclude those suggested by the manufacturer or indicated in the art asbeing appropriate for the enzyme performing the manipulation.

Just as with homology, it is understood that there are a variety ofmethods herein disclosed for determining the level of hybridizationbetween two nucleic acid molecules. It is understood that these methodsand conditions may provide different percentages of hybridizationbetween two nucleic acid molecules, but unless otherwise indicatedmeeting the parameters of any of the methods would be sufficient. Forexample if 80% hybridization was required and as long as hybridizationoccurs within the required parameters in any one of these methods it isconsidered disclosed herein.

It is understood that those of skill in the art understand that if acomposition or method meets any one of these criteria for determininghybridization either collectively or singly it is a composition ormethod that is disclosed herein.

c) Nucleotides and Related Molecules

These discussion of nucleotides and nucleotide related molecules are notmeant to be limiting, but rather are exemplary of the wide range ofcompositions that are related to nucleotide molecules.

A nucleotide is a molecule that contains a base moiety, a sugar moietyand a phosphate moiety. Nucleotides can be linked together through theirphosphate moieties and sugar moieties creating an internucleosidelinkage. The base moiety of a nucleotide can be adenin-9-yl (A),cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T).The sugar moiety of a nucleotide is a ribose or a deoxyribose. Thephosphate moiety of a nucleotide is pentavalent phosphate. Annon-limiting example of a nucleotide would be 3′-AMF (3′-adenosinemonophosphate) or 5′-GMP (5′-guanosine monophosphate).

It is understood when the terms “nucleotide” or “nucleotides” are usedthat nucleotide analogs, nucleotides substitutions, any other nucleotiderelated molecules are contemplated unless specifically indicated to thecontrary.

A nucleotide analog is a nucleotide which contains some type ofmodification to either the base, sugar, or phosphate moieties.Modifications to the base moiety would include natural and syntheticmodifications of A, C, G, and T/U as well as different purine orpyrimidine bases, such as uracil-5-yl (.psi.), hypoxanthin-9-yl (I), and2-aminoadenin-9-yl. A modified base includes but is not limited to5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional basemodifications can be found for example in U.S. Pat. No. 3,687,808,Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and Sanghvi, Y. S., Chapter 15, Antisense Research andApplications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRCPress, 1993. Certain nucleotide analogs, such as 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine can increase the stability of duplex formation. Oftenbase modifications can be combined with for example a sugarmodification, such as 2′-O-methoxyethyl, to achieve unique propertiessuch as increased duplex stability. There are numerous United StatesPatent Nos. such as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, which detail and describe a range of basemodifications. Each of these patents is herein incorporated byreference.

Nucleotide analogs can also include modifications of the sugar moiety.Modifications to the sugar moiety would include natural modifications ofthe ribose and deoxy ribose as well as synthetic modifications. Sugarmodifications include but are not limited to the following modificationsat the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-,S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the allyl, alkenyl andalkynyl may be substituted or unsubstituted C₁ to C₁₀, alkyl or C₂ toC₁₀ alkenyl and alkynyl. 2′ sugar modifications also include but are notlimited to —O[(CH₂)_(n)O]_(m) CH₃, —O(CH₂)_(n) OCH₃, —O(CH₂)_(n) NH₂,—O(CH₂)_(n) CH₃, —O(CH₂)_(n)—ONH₂, and —O(CH₂)_(n)ON[(CH₂)_(n) CH₃)]₂,where n and m are from 1 to about 10.

Other modifications at the 2′ position include but are not limited to:C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacolcinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. Similar modifications mayalso be made at other positions on the sugar, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide. Modifiedsugars would also include those that contain modifications at thebridging ring oxygen, such as CH₂ and S. Nucleotide sugar analogs mayalso have sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. There are numerous United States patents thatteach the preparation of such modified sugar structures such as U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference in its entirety.

Nucleotide analogs can also be modified at the phosphate moiety.Modified phosphate moieties include but are not limited to those thatcan be modified so that the linkage between two nucleotides contains aphosphorothioate, chiral phosphorothioate, phosphorodithioate,phosphotriester, aminoalkylphosphotriester, methyl and other alkylphosphonates including 3′-alkylene phosphonate and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andamninoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates. It is understood that these phosphate or modifiedphosphate linkage between two nucleotides can be through a 3′-5′ linkageor a 2′-5′ linkage, and the linkage can contain inverted polarity suchas 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and freeacid forms are also included. Numerous United States patents teach howto make and use nucleotides containing modified phosphates and includebut are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is hereinincorporated by reference.

It is understood that nucleotide analogs need only contain a singlemodification, but may also contain multiple modifications within one ofthe moieties or between different moieties.

Nucleotide substitutes are molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize nucleic acids in a Watson-Crick or Hoogsteen manner,but which are linked together through a moiety other than a phosphatemoiety. Nucleotide substitutes are able to conform to a double helixtype structure when interacting with the appropriate target nucleicacid.

Nucleotide substitutes are nucleotides or nucleotide analogs that havehad the phosphate moiety and/or sugar moieties replaced. Nucleotidesubstitutes do not contain a standard phosphorus atom. Substitutes forthe phosphate can be for example, short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts. Numerous United States patents disclosehow to make and use these types of phosphate replacements and includebut are not limited to U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439,each of which is herein incorporated by reference.

It is also understood in a nucleotide substitute that both the sugar andthe phosphate moieties of the nucleotide can be replaced, by for examplean amide type linkage (aminoethylglycine) (PNA). U.S. Pat. Nos.5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNAmolecules, each of which is herein incorporated by reference. (See alsoNielsen et al., Science, 1991, 254, 1497-1500).

It is also possible to link other types of molecules (conjugates) tonucleotides or nucleotide analogs to enhance for example, cellularuptake. Conjugates can be chemically linked to the nucleotide ornucleotide analogs. Such conjugates include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al, Proc. Natl.Acad. Sci. USA, 1989,

86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let.,1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharanet al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al.,Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chainManoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexyla-mino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937. Numerous United States patents teach thepreparation of such conjugates and include, but are not limited to U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,each of which is herein incorporated by reference.

A Watson-Crick interaction is at least one interaction with theWatson-Crick face of a nucleotide, nucleotide analog, or nucleotidesubstitute. The Watson-Crick face of a nucleotide, nucleotide analog, ornucleotide substitute includes the C2, N1, and C6 positions of a purinebased nucleotide, nucleotide analog, or nucleotide substitute and theC2, N3, C4 positions of a pyrimidine based nucleotide, nucleotideanalog, or nucleotide substitute.

A Hoogsteen interaction is the interaction that takes place on theHoogsteen face of a nucleotide or nucleotide analog, which is exposed inthe major groove of duplex DNA. The Hoogsteen face includes the N7position and reactive groups (NH2 or O) at the C6 position of purinenucleotides.

Nucleic acids are typically made up of nucleotides, nucleotide analogs,or nucleotide substitutions, or combinations thereof.

d) Sequences

Disclosed are nucleic acids and peptides. Certain disclosed nucleicacids, encode peptides that have a particular function, such as bindingTRAP, increasing osteoblast binding to TRAP and to osteoclast and/orosteoclast lacuna, or inhibiting osteoblast binding to osteoclastlacuna, in for example a competitive binding reaction with TRAP orosteoclasts or osteoclast lacuna. Other disclosed nucleic acids canthemselves either inhibit or affect the interactions between osteoblastsand osteoclast lacuna or TRAP, for example, such as aptamers, or thatcan effect the expression of various nucleic acids, such as antisensemolecules. Other nucleic acids that are disclosed are related to genesthat produce proteins expressed in either osteoclasts or osteoblasts,for example.

For example, there are a variety of sequences related to the GPC4 gene(nucleic acid is SEQ ID NO:37, peptide sequence is SEQ ID NO:38) havingthe following Genbank Accession Number: XM_(—)029542, and the TRIP gene(nucleic acid is SEQ ID NO:39 and peptide is SEQ ID NO:40) havingGenbank accession number U36764 these sequences and others are hereinincorporated by reference in their entireties as well as for individualsubsequences contained therein.

One particular sequence, set forth in SEQ ID NO:37 and having Genbankaccession number XM_(—)029542 is used herein, as an example, toexemplify the disclosed compositions and methods. It is understood thatthe description related to this sequence is applicable to any sequencerelated to GPC4 or TRIP or any other disclosed sequence unlessspecifically indicated otherwise. Those of skill in the art understandhow to resolve sequence discrepancies and differences and to adjust thecompositions and methods relating to a particular sequence to otherrelated sequences (i.e. sequences of TRIP). Primers and/or probes can bedesigned for any GPC4 or TRI sequence or sequence disclosed herein,given the information disclosed herein and known in the art.

e) TRAP Binding Region GPC4

The GPC4 gene encodes a protein that contains a TRAP binding region,which is amino acids 252-263 of SEQ ID NO:38. This region has been shownto have a high degree of homology to clone 5, which is SEQ ID NO:23. Itis understood that the region defined by amino acids 252-263 of the SEQID NO:38 represent one variant of a TRAP binding region, and that thereare other variants which will function in binding TRAP. For example,this sequence indicates that there is a 5 amino acid stretch that is100% conserved, amino acids 259-263 of SEQ ID NO:38. Thus, disclosed areisolated compositions that comprise amino acids 259-263 of SEQ ID NO:38,which function to bind TRAP or compositions which encode these aminoacids. Also disclosed are isolated compositions that comprise aminoacids 259-263 of SEQ ID NO:38, which function to bind TRAP orcompositions which encode these amino acids which are non-naturalproteins or nucleic acids. Also disclosed are compositions having thelevel of homology observed between SEQ ID NO:23 and SEQ ID NO:38.Furthermore, it is understood that there are regions for attachment tothe cellular membrane. Variants, as discussed herein, are considereddisclosed and described.

f) Primers and Probes

Disclosed are compositions including primers and probes, which arecapable of interacting with the GPC4, TRIP, and TRAP genes or mRNAs ortheir complements or any other nucleic acid as disclosed herein. Incertain embodiments the primers are used to support DNA amplificationreactions. Typically the primers will be capable of being extended in asequence specific manner. Extension of a primer in a sequence specificmanner includes any methods wherein the sequence and/or composition ofthe nucleic acid molecule to which the primer is hybridized or otherwiseassociated directs or influences the composition or sequence of theproduct produced by the extension of the primer. Extension of the primerin a sequence specific manner therefore includes, but is not limited to,PCR, DNA sequencing, DNA extension, DNA polymerization, RNAtranscription, or reverse transcription. Techniques and conditions thatamplify the primer in a sequence specific manner are preferred. Incertain embodiments the primers are used for the DNA amplificationreactions, such as PCR or direct sequencing. It is understood that incertain embodiments the primers can also be extended using non-enzymatictechniques, where for example, the nucleotides or oligonucleotides usedto extend the primer are modified such that they will chemically reactto extend the primer in a sequence specific manner.

The size of the primers or probes for interaction with, for example,GPC4 or TRIP or TRAP genes, mRNA or their complements in certainembodiments can be any size that supports the desired enzymaticmanipulation of the primer, such as DNA amplification or the simplehybridization of the probe or primer. A typical GPC4 or TRIP or TRAPprimer or probe, for example, would be at least about 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500,1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides long.

In other embodiments a GPC4 or TRIP or TRAP primer or probe, forexample, can be less than or equal to about 6, 7, 8, 9, 10, 11, 12 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150,175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750,2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides long.

The primers for the GPC4 or TRIP or TRAP typically will be used toproduce an amplified DNA product. In general, typically the size of theproduct will be such that the size can be accurately determined towithin 3, or 2 or 1 nucleotides.

In certain embodiments this product is at least about 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000nucleotides long.

In other embodiments the product is less than or equal to about 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000,3500, or 4000 nucleotides long.

g) Functional Nucleic Acids

Disclosed are compositions and methods that can involve functionalnucleic acids, such as nucleic acids that affect the mRNA or interactwith the mRNA of for example, GPC4, TRIP or TRAP. Other functionalnucleic acids and methods of using them, may affect or interact with thegenes of GPC4, TRIP, or TRAP. Other functional nucleic acids and methodsof using them, may affect or interact with the function of the geneproducts of GPC4, TRIP, or TRAP. Also disclosed are methods of isolatingand identifying the functional nucleic acids disclosed herein.

Functional nucleic acids are nucleic acid molecules that have a specificfunction, such as binding a target molecule or catalyzing a specificreaction. Functional nucleic acid molecules can be divided into thefollowing categories, which are not meant to be limiting. For example,functional nucleic acids include antisense molecules, aptamers,ribozymes, triplex forming molecules, and external guide sequences. Thefunctional nucleic acid molecules can act as affectors, inhibitors,modulators, and stimulators of a specific activity possessed by a targetmolecule, or the functional nucleic acid molecules can possess a de novoactivity independent of any other molecules.

Functional nucleic acid molecules can interact with any macromolecule,such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functionalnucleic acids can interact with the mRNA of GPC4, TRIP, or TRAP or thegenomic DNA of GPC4, TRIP, or TRAP or they can interact with the GPC4,TRIP, or TRAP polypeptide. Often functional nucleic acids are designedto interact with other nucleic acids based on sequence homology betweenthe target molecule and the functional nucleic acid molecule. In othersituations, the specific recognition between the functional nucleic acidmolecule and the target molecule is not based on sequence homologybetween the functional nucleic acid molecule and the target molecule,but rather is based on the formation of tertiary structure that allowsspecific recognition to take place.

Antisense molecules are designed to interact with a target nucleic acidmolecule through either canonical or non-canonical base pairing. Theinteraction of the antisense molecule and the target molecule isdesigned to promote the destruction of the target molecule through, forexample, RNAseH mediated RNA-DNA hybrid degradation. Alternatively theantisense molecule is designed to interrupt a processing function thatnormally would take place on the target molecule, such as transcriptionor replication. Antisense molecules can be designed based on thesequence of the target molecule. Numerous methods for optimization ofantisense efficiency by finding the most accessible regions of thetarget molecule exist. Exemplary methods would be in vitro selectionexperiments and DNA modification studies using DMS and DEPC. It ispreferred that antisense molecules bind the target molecule with adissociation constant (K_(d)) less than 10⁻⁶. It is more preferred thatantisense molecules bind with a K_(d) less than 10⁻⁸. It is also morepreferred that the antisense molecules bind the target molecule with aK_(d) less than 10⁻¹⁰. It is also preferred that the antisense moleculesbind the target molecule with a K_(d) less than 10⁻¹². A representativesample of methods and techniques which aid in the design and use ofantisense molecules can be found in the following non-limiting list ofU.S. Pat. Nos. 5,135,917, 5,294,533, 5,627,158, 5,641,754, 5,691,317,5,780,607, 5,786,138, 5,849,903, 5,856,103, 5,919,772, 5,955,590,5,990,088, 5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522,6,017,898, 6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004,6,046,319, and 6,057,437.

Antisense molecules that interact with the mRNA of GPC4 and TRIP or TRAPare disclosed. For example, antisense molecules to GPC4 are provided inKleeff J. Wildi S. Kumbasar A. Friess H. Lander A D. Korc M. Stabletransfection of a glypican antisense construct decreases tumorigenicityin PANC-1 pancreatic carcinoma cells. Pancreas. 19(3):281-8,1999 andKleeff J. Wildi S. Kumbasar A. Friess H. Lander A D. Korc M. Stabletransfection of a glypican-1 antisense construct decreasestumorigenicity in PANC-1 pancreatic carcinoma cells. Pancreas.19(3):281-8,1999 which are herein incorporated by reference at least forthe material related to antisense of GPC4.

Aptamers are molecules that interact with a target molecule, preferablyin a specific way. Typically aptamers are small nucleic acids rangingfrom 15-50 bases in length that fold into defined secondary and tertiarystructures, such as stem-loops or G-quartets. Aptamers can bind smallmolecules, such as ATP (U.S. Pat. No. 5,631,146) and theophiline (U.S.Pat. No. 5,580,737), as well as large molecules, such as reversetranscriptase (U.S. Pat. No. 5,786,462) and thrombin (U.S. Pat. No.5,543,293). Aptamers can bind very tightly with k_(d)s from the targetmolecule of less than 10⁻¹² M. It is preferred that the aptamers bindthe target molecule with a K_(d) less than 10⁻⁶. It is more preferredthat the aptamers bind the target molecule with a K_(d) less than 10⁻⁸.It is also more preferred that the aptamers bind the target moleculewith a K_(d) less than 10⁻¹⁰. It is also preferred that the aptamersbind the target molecule with a K_(d) less than 10⁻¹². Aptamers can bindthe target molecule with a very high degree of specificity. For example,aptamers have been isolated that have greater than a 10000 folddifference in binding affinities between the target molecule and anothermolecule that differ at only a single position on the molecule (U.S.Pat. No. 5,543,293). It is preferred that the aptamer have a K_(d) withthe target molecule at least 10 fold lower than the K_(d) with abackground binding molecule. It is more preferred that the aptamer havea K_(d) with the target molecule at least 100 fold lower than the K_(d)with a background binding molecule. It is more preferred that theaptamer have a K_(d) with the target molecule at least 1000 fold lowerthan the K_(d) with a background binding molecule. It is preferred thatthe aptamer have a K_(d) with the target molecule at least 10000 foldlower than the K_(d) with a background binding molecule. It is preferredwhen doing the comparison for a polypeptide for example, that thebackground molecule be a different polypeptide. For example, whendetermining the specificity of GPC4, TRIP, or TRAP aptamers, thebackground protein could be bovine serum albumin. Representativeexamples of how to make and use aptamers to bind a variety of differenttarget molecules can be found in the following non-limiting list of U.S.Pat. Nos. 5,476,766, 5,503,978, 5,631,146, 5,731,424, 5,780,228,5,792,613, 5,795,721, 5,846,713, 5,858,660, 5,861,254, 5,864,026,5,869,641, 5,958,691, 6,001,988, 6,011,020, 6,013,443, 6,020,130,6,028,186, 6,030,776, and 6,051,698.

Ribozymes are nucleic acid molecules that are capable of catalyzing achemical reaction, either intramolecularly or intermolecularly.Ribozymes are thus catalytic nucleic acid. It is preferred that theribozymes catalyze intermolecular reactions. There are a number ofdifferent types of ribozymes that catalyze nuclease or nucleic acidpolymerase type reactions which are based on ribozymes found in naturalsystems, such as hammerhead ribozymes, (for example, but not limited tothe following U.S. Pat. Nos. 5,334,711, 5,436,330, 5,616,466, 5,633,133,5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288,5,891,683, 5,891,684, 5,985,621, 5,989,908, 5,998,193, 5,998,203, WO9858058 by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO9718312 by Ludwig and Sproat) hairpin ribozymes (for example, but notlimited to the following U.S. Pat. Nos. 5,631,115, 5,646,031, 5,683,902,5,712,384, 5,856,188, 5,866,701, 5,869,339, and 6,022,962), andtetrahymena ribozymes (for example, but not limited to the followingU.S. Pat. Nos. 5,595,873 and 5,652,107). There are also a number ofribozymes that are not found in natural systems, but which have beenengineered to catalyze specific reactions de novo (for example, but notlimited to the following U.S. Pat. Nos. 5,580,967, 5,688,670, 5,807,718,and 5,910,408). Preferred ribozymes cleave RNA or DNA substrates, andmore preferably cleave RNA substrates. Ribozymes typically cleavenucleic acid substrates through recognition and binding of the targetsubstrate with subsequent cleavage. This recognition is often basedmostly on canonical or non-canonical base pair interactions. Thisproperty makes ribozymes particularly good candidates for targetspecific cleavage of nucleic acids because recognition of the targetsubstrate is based on the target substrates sequence. Representativeexamples of how to make and use ribozymes to catalyze a variety ofdifferent reactions can be found in the following non-limiting list ofU.S. Pat. Nos. 5,646,042, 5,693,535, 5,731,295, 5,811,300, 5,837,855,5,869,253, 5,877,021, 5,877,022, 5,972,699, 5,972,704, 5,989,906, and6,017,756.

Triplex forming functional nucleic acid molecules are molecules that caninteract with either double-stranded or single-stranded nucleic acid.When triplex molecules interact with a target region, a structure calleda triplex is formed, in which there are three strands of DNA forming acomplex dependant on both Watson-Crick and Hoogsteen base-pairing.Triplex molecules are preferred because they can bind target regionswith high affinity and specificity. It is preferred that the triplexforming molecules bind the target molecule with a K_(d) less than 10⁻⁶.It is more preferred that the triplex forming molecules bind with aK_(d) less than 10⁻⁸. It is also more preferred that the triplex formingmolecules bind the target molecule with a K_(d) less than 10⁻¹⁰. It isalso preferred that the triplex forming molecules bind the targetmolecule with a K_(d) less than 10⁻¹². Representative examples of how tomake and use triplex forming molecules to bind a variety of differenttarget molecules can be found in the following non-limiting list of U.S.Pat. Nos. 5,176,996, 5,645,985, 5,650,316, 5,683,874, 5,693,773,5,834,185, 5,869,246, 5,874,566, and 5,962,426.

External guide sequences (EGSs) are molecules that bind a target nucleicacid molecule forming a complex, and this complex is recognized by RNaseP, which cleaves the target molecule. EGSs can be designed tospecifically target a RNA molecule of choice. RNAse P aids in processingtransfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited tocleave virtually any RNA sequence by using an EGS that causes the targetRNA:EGS complex to mimic the natural tRNA substrate. (WO 92/03566 byYale, and Forster and Altman, Science 238:407-409 (1990)).

Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can beutilized to cleave desired targets within eukarotic cells. (Yuan et al.,Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992); WO 93/22434 by Yale; WO95/24489 by Yale; Yuan and Altman, EMBO J 14:159-168 (1995), and Carraraet al., Proc. Natl. Acad. Sci. (USA) 92:2627-2631 (1995)).Representative examples of how to make and use EGS molecules tofacilitate cleavage of a variety of different target molecules be foundin the following non-limiting list of U.S. Pat. Nos. 5,168,053,5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877,162

5. Peptides

Disclosed are isolated peptides that bind TRAP. For example, disclosedare isolated peptides set forth in SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or SEQ IDNO:36.

Also disclosed are isolated peptides comprising at least 80% identity toa peptide set forth in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36.

Also disclosed are isolated peptides comprising at least 80% identity toa peptide set forth in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:3 1, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36,wherein any change from the SEQ ID Nos:19-36 are conservative changes.

Also disclosed are isolated peptides, wherein the peptide binds anosteoclast cell and isolated peptides, wherein the peptides bindsosteoclast lacuna and isolated peptides, wherein the peptide binds alysosomal protein expressed in osteoclasts and isolated peptides,wherein the lysosomal protein is tartrate resistant acid phosphatase.

Disclosed are isolated peptides, wherein the peptides bind with a K_(d)less than or equal to 10⁻⁵, wherein the peptides bind with a K_(d) lessthan or equal to 10⁻⁶, wherein the peptides bind with a K_(d) less thanor equal to 10⁻⁷, wherein the peptides bind with a K_(d) less than orequal to 10⁻⁸, wherein the peptides bind with a K_(d) less than or equalto 10⁻⁹, wherein the peptides bind with a K_(d) less than or equal to10⁻¹⁰, wherein the peptides bind with a K_(d) less than or equal to10⁻¹¹, wherein the peptides bind with a K_(d) less than or equal to10⁻¹².

Also disclosed are cells comprising any of the non-naturally occurringdisclosed peptides. Non-naturally occurring means occurring in a cell ina way that has been affected by a recombinant molecular biologytechnique or delivery of the composition to the cell.

Also disclosed are animals comprising any of the non-naturally occurringdisclosed peptides.

Disclosed are pharmaceutical compositions comprising any of thedisclosed peptides and a pharmaceutically acceptable carrier.

Disclosed are isolated peptides, wherein the peptide inhibits thebinding of an osteoblast to an ostcoclast lacuna.

Also disclosed are isolated peptides, wherein the peptides have a K_(d)of inhibition less than or equal to 10⁻⁵ and, wherein the peptides havea K_(d) of inhibition less than or equal to 10⁻⁶ and, wherein thepeptides have a K_(d) of inhibition less than or equal to 10⁻⁷ and,wherein the peptides have a K_(d) of inhibition less than or equal to10⁻⁸ and, wherein the peptides have a K_(d) of inhibition less than orequal to 10⁻⁹ and, wherein the peptides have a K_(d) of inhibition lessthan or equal to 10⁻¹⁰ and, wherein the peptides have a K_(d) ofinhibition less than or equal to 10⁻¹¹ and, wherein the peptides have aK_(d) of inhibition less than or equal to 10⁻¹².

Also disclosed are isolated peptides, wherein the identity is at least81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99%.

a) Protein Variants

As discussed herein there are numerous variants of the protein andpolypeptides set forth in SEQ ID NOs: 19-36, 38, 40, and 42 that areknown and herein contemplated. In addition, to the naturally occurringfuictional variants in SEQ ID NOs: 38, 40, and 42 that are homologvariants (varying species for example) there are non-naturally occurringvariant of the in SEQ ID NOs: 19-36, 38, 40, and 42 proteins which alsofinction in the disclosed methods and compositions. Protein variants andderivatives are well understood to those of skill in the art and caninvolve amino acid sequence modifications. For example, amino acidsequence modifications typically fall into one or more of three classes:substitutional, insertional or deletional variants. Insertions includeamino and/or carboxyl terminal fusions as well as intrasequenceinsertions of single or multiple amino acid residues. Insertionsordinarily will be smaller insertions than those of amino or carboxylterminal fusions, for example, on the order of one to four residues.Immunogenic fusion protein derivatives, such as those described in theexamples, are made by fusing a polypeptide sufficiently large to conferimmunogenicity to the target sequence by cross-linking in vitro or byrecombinant cell culture transformed with DNA encoding the fusion.Deletions are characterized by the removal of one or more amino acidresidues from the protein sequence. Typically, no more than about from 2to 6 residues are deleted at any one site within the protein molecule.These variants ordinarily are prepared by site specific mutagenesis ofnucleotides in the DNA encoding the protein, thereby producing DNAencoding the variant, and thereafter expressing the DNA in recombinantcell culture. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, forexample M13 primer mutagenesis and PCR mutagenesis. Amino acidsubstitutions are typically of single residues, but can occur at anumber of different locations at once; insertions usually will be on theorder of about from 1 to 10 amino acid residues; and deletions willrange about from 1 to 30 residues. Deletions or insertions preferablyare made in adjacent pairs, i.e. a deletion of 2 residues or insertionof 2 residues. Substitutions, deletions, insertions or any combinationthereof may be combined to arrive at a final construct. The mutationsmust not place the sequence out of reading frame and preferably will notcreate complementary regions that could produce secondary mRNAstructure. Substitutional variants are those in which at least oneresidue has been removed and a different residue inserted in its place.Such substitutions can generally be made as conservative substitutionswhich generally are made in accordance with the following Tables 1 and2. TABLE 1 Amino Acid Abbreviations Amino Acid Abbreviations alanine AlaA allosoleucine AIle arginine Arg R asparagine Asn N aspartic acid Asp Dcysteine Cys C glutamic acid Glu E glutamine Gln K glycine Gly Ghistidine His H isolelucine Ile I leucine Leu L lysine Lys Kphenylalanine Phe F proline Pro P pyroglutamic acid pGlu serine Ser Sthreonine Thr T tyrosine Tyr Y tryptophan Trp W valine Val V

TABLE 2 Amino Acid Substitutions Exemplary Conservative Substitutions,others Original Residue are known in the art. Ala ser Arg lys, gln Asngln; his Asp glu Cys ser Gln asn, lys Glu asp Gly pro His asn; gln Ileleu; val Leu ile; val Lys arg; gln; Met Leu; ile Phe met; leu; tyr Serthr Thr ser Trp tyr Tyr trp; phe Val ile; leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table2, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in the proteinproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine, in this case, (e) by increasing the number of sites forsulfation and/or glycosylation.

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser,Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variationsof each explicitly disclosed sequence are included within the mosaicpolypeptides provided herein.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, isaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the o-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco pp 79-86[1983]), acetylation of the N-terminal amine and, in some instances,amidation of the C-terminal carboxyl.

It is understood that one way to define the variants and derivatives ofthe disclosed proteins herein is through defining the variants andderivatives in terms of homology/identity to specific known sequences.For example, SEQ ID NO:23 sets forth a particular sequence of clone 5and SEQ ID NO:38 sets forth a particular sequence of a GPC4 protein.Specifically disclosed are variants of these and other proteins hereindisclosed which have at least 40 or 50 or 60 or 70% or 75% or 80% or 85%or 90% or 95% homology to the stated sequence. Those of skill in the artreadily understand how to determine the homology of two proteins. Forexample, the homology can be calculated after aligning the two sequencesso that the homology is at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment.

It is understood that the description of conservative mutations andhomology can be combined together in any combination, such asembodiments that have at least 40% homology to a particular sequencewherein the variants are conservative mutations.

As this specification discusses various proteins and protein sequencesit is understood that the nucleic acids that can encode those proteinsequences are also disclosed. This would include all degeneratesequences related to a specific protein sequence, i.e. all nucleic acidshaving a sequence that encodes one particular protein sequence as wellas all nucleic acids, including degenerate nucleic acids, encoding thedisclosed variants and derivatives of the protein sequences. Thus, whileeach particular nucleic acid sequence may not be written out herein, itis understood that each and every sequence is in fact disclosed anddescribed herein through the disclosed protein sequence. For example,one of the many nucleic acid sequences that can encode the proteinsequence set forth in SEQ ID NO:23 is set forth in SEQ ID NO:5. Anothernucleic acid sequence that encodes the same protein sequence set forthin SEQ ID NO:23 is set forth in SEQ ID NO:43. In addition, for example,a disclosed conservative derivative of SEQ ID NO:23 is shown in SEQ IDNO: 44, where the valine (V) at position 12 is changed to a isoleucine(I). It is understood that for this mutation all of the nucleic acidsequences that encode this particular derivative of SEQ ID NO:23 arealso disclosed including for example SEQ ID NO:45 and SEQ ID NO:46 whichset forth two of the degenerate nucleic acid sequences that encode theparticular polypeptide set forth in SEQ ID NO:44. For example, one ofthe many nucleic acid sequences that can encode the protein sequence setforth in SEQ ID NO:38 is set forth in SEQ ID NO:37. Another nucleic acidsequence that encodes the same protein sequence set forth in SEQ IDNO:38 is set forth in SEQ ID NO:47. In addition, for example, adisclosed conservative derivative of SEQ ID NO:38 is shown in SEQ ID NO:48, where the G at position 5 is changed to an A. It is understood thatfor this mutation all of the nucleic acid sequences that encode thisparticular derivative of SEQ ID NO:48 are also disclosed including forexample SEQ ID NO:49 and SEQ ID NO:50 which set forth two of thedegenerate nucleic acid sequences that encode the particular polypeptideset forth in SEQ ID NO:48. It is also understood that while no aminoacid sequence indicates what particular DNA sequence encodes thatprotein within an organism, where particular variants of a disclosedprotein are disclosed herein, the known nucleic acid sequence thatencodes that protein in the particular organism from which that proteinarises is also known and herein disclosed and described.

b) GPC4

Glypican 4 is one member of a close family of heparan sulfateproteoglycan-containing plasma membrane receptors found on fibroblasts,periodontal ligament cells and mesenchymal and marrow stem cells(Worapamom W, Li H, Haas H R, Pujic Z, Ghrjes A A, Bartold P M (2000)Cell surface proteoglycan expression in human periodontal cells.Connective Tiss. Res. 41: 57-68 and Siebertz B, Stocker G, Drzeniek Z,Handt S, Just U, Haubeck H D (1999) Expression of glypican-4 inhaematopoietic-progenitor and bone marrow stromal cells. Biochemical. J.344:937-43). These receptors have been implicated in BMP signaling inDrosophila and as cytokine presenting receptors in bone marrow cells.Mutations in the glypican family lead to a syndrome known atSimpson-Golabi-Behmel syndrome (24.Pilia G, Huges-Benzi R M, MacKenzieA, Baybayan P, Chen E Y, Huber R, Neri G, Cao A, Forabosco A,Schlessinger D (1996) Mutatations in GPC, a glypican gene, cause theSimpson-Golabi-Behmel overgrowth syndrome. Nature Genetics 12:241-247).Mice with this syndrome demonstrate a varied phenotype, however, the onecommon feature is skeletal abnormalities. The phenotype is shortenedlimbs, malformed trabecular architecture and a compressed rib cage.Tooth development may also be affectes.

c) TRAP

TRAP is a lysomsomal protein that is expressed in osteoclast cells. TRAPis exported from the osteoclast cell and resides between the membrane ofthe osteoclast cell and the bone surface which is undergoing resorption.

There are upward of 20 characterized active lysosomal enzymes thatparticipate in osteoclastic bone resorption which are discussed inKremer M, Judd J, Rifkin B, Auszmann J, Oursler M J (1995) Estrogenmodulation of osteoclast lysosomal enzyme secretion. Journal of CellularBiochemistry. 57:271-9; Vaes G, Delaisse J M, Eeckhout Y (1992) Relativeroles of collagenase and lysosomal cysteine-proteinases in boneresorption. Matrix Supplement. 1:383-8; Sasaki T, Ueno-Matsuda E (1993)Cysteine-proteinase localization in osteoclasts: an immunocytochemicalstudy. Cell & Tissue Research. 27:177-9; Ohsawa Y, Nitatori T, HiguchiS, Kominami E, Uchiyama Y (1993) Lysosomal cysteine and asparticproteinases, acid phosphatase, and an endogenous cysteine proteinaseinhibitor, cystatin-beta, in rat osteoclasts. Journal of Histochemistry& Cytochemistry. 41:1075-83; Baron R(1989) Molecular mechanisms of boneresorption by the osteoclast. Anatomical Record. 224:317-24; KarhukorpiE K, Vihko P, Vaananen K (1992) A difference in the enzyme contents ofresorption lacunae and secondary lysosomes of osteoclasts. ActaHistochemica. 92:1-11; Delaisse J M, Eeckhout Y, Vaes G (1985)Bisphosphonates and bone resorption: effects on collagenase andlysosomal enzyme excretion. Life Sciences. 37:2291-6; Baron R, Neff L,Tran Van P, Nefussi J R, Vignery A (1986) Kinetic and cytochemicalidentification of osteoclast precursors and their differentiation intomultinucleated osteoclasts. American Journal of Pathology. 122:363-78;Ash P, Loutit J F, Townsend K M (1980) Giant lysosomes, a cytoplasmicmarker in osteoclasts of beige mice. Journal of Pathology. 130:237-45;Lorenzo J A, Holtrop M E, Raisz L G (1984) Effects of phosphate oncalcium release, lysosomal enzyme activity in the medium, and osteoclastmorphometry in cultured fetal rat bones. Metabolic Bone Disease &Related Research. 5:187-90 which are herein incorporated by reference atleast for material related to their cognate lysozymes.

Type V tartrate resistant acid phosphatase (TRAP) is present in a numberof cell types, the most abundant of which may be active osteoclasts. Infact, TRAP is the acid hydrolase that has become a standardized markerfor identification of this cell type (Hayman A R, et al., J. of Anatomy.196:433-41 (2000); Tiffee J C, Aufdemorte T B,. Journal of Oral &Maxillofacial Surgery. 55:1108-12 (1997)). Moreover, we have known formany years that there is a polarized secretion of TRAP and otherlysosomal enzymes by the osteoclast toward the bone surface (Baron R, etal., et al., Journal of Cell Biology. 106:1863-72.) and that theseenzymes can be detected deep within bone matrix and at sites ofresorption after the osteoclast has left the lacuna (Wergedal J E,Baylink D J, Journal of Histochemistry & Cytochemistry. 17:799-806(1969); Yamamoto T, Nagai H Journal of Bone & Mineral Research.7:1267-73 (1992)).

Maintenance of trabecular bone architecture is necessary to withstandmechanical forces and stresses and to resist skeletal fractures.Mechanical properties of bone (reviewed in Bikle D D, Halloran B P,Journal of Bone & Mineral Metabolism. 17:233-44 (1999), micro-cracks(Hirano T. et al., Bone. 27(1):13-20, 2000.), endocrine regulators(reviewed in Seeman E. Delmas P D., Trends in Endocrinology &Metabolism. 12(7):281-3, 2001) and remodeling events are all likelyfactors involved in controlling the amount of bone at any particularskeletal site. TRAP appears to participate in the spatial orientation ofwhere bone is formed during skeletal remodeling.

The experiments reported herein were performed with two sources of TRAP.One was from a purified protein extraction. This molecule wouldpresumably be modified by post translational reactions. The secondsource of TRAP was material created as a GST fusion protein. Bothsources of TRAP behaved similarly in the binding and activity assays.This finding argues that it is likely to be a specific amino acidsequence in the TRAP molecule that binds to TRIP-1 rather than apost-translationally added carbohydrate moiety.

A recombinant GST-TRAP fusion protein was made and shown to functionlike glycosylated TRAP. This recombinant protein is a non-glycosylprotein because prokaryotic cells don't have this post translationmodification system. The purified TRAP proteins from mammalian sourceusually are highly glycosylated. Most of the polysaccharide moiety ismannose, mannose-6-phosphate and sialic acid.

TRAP is the molecule to induce ROS 17/2.8 and D14 osteoblast cellsapoptosis but not from impurities, and the post-translation modificationof TRAP is not required for the apoptosis effect.

d) TRIP

TRIP-1 has been characterized as a modulator of the TGFβ response (Choy,L. and Derynck, R., J. Biol. Chem. 273: 31455-31462 (1998)). It is aWD40 repeat-containing protein that is a phosphorylation substrate forthe type II TGFβ receptor. TRIP-1, when phosphorylated, repressesTGFβ-driven reporter activity from the plasminogen activator inhibitor-1(PAI-1) promoter but has no effect on the TGFβ-driven cyclin A promoter(Choy, L. and Derynck, R., J. Biol. Chem. 273: 31455-31462 (1998)). Thedisclosed data indicate that when TRAP associates with TRIP-1, there isan activation of TGFβ signaling, which is consistent with theTRAP/TRIP-1 association blocking phosphorylation and allowing a fullexpression of the TGFβ signaling pathway. TRIP-1 homologs have also beenidentified in plants. Their function in these systems is not known,however, speculation that they may be involved in cell cycle activity issupported by their similarity to a translation initiation factor (Jiang,J. and Clouse, S. D., The Plant Journal 26:, 35-45 (2001)).

Mice deficient in osteoclast TRAP demonstrate skeletal abnormalities(Hayman A R. et al., Development. 122(10):3151-62, (1996)). As expected,TRAP-null mice have a compromised ability to resorb bone throughdefective osteoclast activity. This is manifested as a mildosteopetrosis, i.e. skeletal density is slightly increased. However, acloser examination of the bone reveals a haphazard and disorganizedmicro-architecture. Additional evidence that removal of TRAP and othersite-directing signals leads to inappropriate bone formation has beenfound at sites of inflammation and infection in bone, such as inperiodontal disease (33). Bacterial and inflammatory cell activity atalveolar bone sites could very well destroy site-directing signals andprevent normal osteoblast function.

TRIP-1 has been cloned and characterized. TRIP-I is a negative regulatorin the TGF-β signaling pathway. Disclosed herein TRIP-I function wastested in the chick sternal chondrocyte and osteoblast. TRIP-1 has theopposite effect for the TGF-β signaling pathway between these two cells.In check sternal chondrocyte, TRIP-1 is a negative TGF-β signalingregulator. It can diminish almost 60% of TGF-β effect with P3TP-luc as areporter. However, in the osteoblast cell lines (SaOS2 and MG63), TRIP-Ican potentate around two folds the TGF-β signaling pathway. TRAP proteincan turn on the TGF-β signaling pathway by interacting with TRIP-1.

TRIP-1 protein has several WD40 domains in its C-terminal region (ChoyL, Derynck R: J Biol Chem 273:31455-62, 1998), Chen R H, et al., Nature377:548-52, (1995)). This WD 40 domain has been show to interact withRas and then turn on the downstream signaling. Plasmid constructs ofTRIP-1 having varied and deleted WD40 regions exist and can be usedherein (Chen R H, et al., Nature 377:548-52, (1995)). Thus, the dataindicate that TRIP-1 turns on Ras or MEKK pathway through interactionwith the WD40 region, and this interaction affects the apoptoticsignaling through the ras/raf pathway.

6. Delivery of the Compositions to Cells

Both nucleic acid and non-nucleic acid compositions can be delivered tocells either in vitro or in vivo. If a nucleic acid specific deliverysystem, is not used, then typically the delivery must at least be in apharmaceutically acceptable carrier, which are discussed herein.

There are a number of compositions and methods which can be used todeliver nucleic acids to cells, either in vitro or in vivo. Thesemethods and compositions can largely be broken down into two classes:viral based delivery systems and non-viral based delivery systems. Forexample, the nucleic acids can be delivered through a number of directdelivery systems such as, electroporation, lipofection, calciumphosphate precipitation, plasmids, viral vectors, viral nucleic acids,phage nucleic acids, phages, cosmids, or via transfer of geneticmaterial in cells or carriers such as cationic liposomes. Appropriatemeans for transfection, including viral vectors, chemical transfectants,or physico-mechanical methods such as electroporation and directdiffusion of DNA, are described by, for example, Wolff, J. A., et al.,Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818,(1991) Such methods are well known in the art and readily adaptable foruse with the compositions and methods described herein. In certaincases, the methods will be modified to specifically function with largeDNA molecules. Further, these methods can be used to target certaindiseases and cell populations by using the targeting characteristics ofthe carrier.

a) Nucleic Acid Based Delivery Systems

Transfer vectors can be any nucleotide construction used to delivergenes into cells (e.g., a plasmid), or as part of a general strategy todeliver genes, e.g., as part of recombinant retrovirus or adenovirus(Ram et al. Cancer Res. 53:83-88, (1993)).

As used herein, plasmid or viral vectors are agents that transport thedisclosed nucleic acids, such as SEQ ID NO:23 or SEQ ID NO: 37(exogenous gene) into the cell without degradation and include apromoter yielding expression of the exogenous gene in the cells intowhich it is delivered. In some embodiments the delivery molecules arederived from either a virus or a retrovirus. Viral vectors are, forexample, Adenovirus, Adeno-associated virus, Herpes virus, Vacciniavirus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis andother RNA viruses, including these viruses with the HIV backbone. Alsopreferred are any viral families, which share the properties of theseviruses, which make them suitable for use as vectors. Retrovirusesinclude Murine Maloney Leukemia virus, MMLV, and retroviruses thatexpress the desirable properties of MMLV as a vector. Retroviral vectorsare able to carry a larger genetic payload, i.e., a transgene or markergene, than other viral vectors, and for this reason are a commonly usedvector. However, they are not as useful in non-proliferating cells.Adenovirus vectors are relatively stable and easy to work with, havehigh titers, and can be delivered in aerosol formulation, and cantransfect non-dividing cells. Pox viral vectors are large and haveseveral sites for inserting genes, they are thermostable and can bestored at room temperature. A preferred embodiment is a viral vector,which has been engineered so as to suppress the immune response of thehost organism, elicited by the viral antigens. Preferred vectors of thistype will carry coding regions for Interleukin 8 or 10.

Viral vectors can have higher transaction (ability to introduce genes)abilities than chemical or physical methods to introduce genes intocells. Typically, viral vectors contain, nonstructural early genes,structural late genes, an RNA polymerase III transcript, invertedterminal repeats necessary for replication and encapsidation, andpromoters to control the transcription and replication of the viralgenome. When engineered as vectors, viruses typically have one or moreof the early genes removed and a gene or gene/promotor cassette isinserted into the viral genome in place of the removed viral DNA.Constructs of this type can carry up to about 8 kb of foreign geneticmaterial. The necessary functions of the removed early genes aretypically supplied by cell lines which have been engineered to expressthe gene products of the early genes in trans.

(1) Retroviral Vectors

A retrovirus is an animal virus belonging to the virus family ofRetroviridae, including any types, subfamilies, genus, or trophisms.Retroviral vectors, in general, are described by Verma, I. M.,Retroviral vectors for gene transfer. In Microbiology-1985, AmericanSociety for Microbiology, pp. 229-232, Washington, (1985), which isincorporated by reference herein. Examples of methods for usingretroviral vectors for gene therapy are described in U.S. Pat. Nos.4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136;and Mulligan, (Science 260:926-932 (1993)); the teachings of which areincorporated herein by reference.

A retrovirus is essentially a package which has packed into it nucleicacid cargo. The nucleic acid cargo carries with it a packaging signal,which ensures that the replicated daughter molecules will be efficientlypackaged within the package coat. In addition to the package signal,there are a number of molecules, which are needed in cis, for thereplication, and packaging of the replicated virus. Typically aretroviral genome, contains the gag, pol, and env genes which areinvolved in the making of the protein coat. It is the gag, pol, and envgenes, which are typically replaced by the foreign DNA that it is to betransferred to the target cell. Retrovirus vectors typically contain apackaging signal for incorporation into the package coat, a sequencewhich signals the start of the gag transcription unit, elementsnecessary for reverse transcription, including a primer binding site tobind the tRNA primer of reverse transcription, terminal repeat sequencesthat guide the switch of RNA strands during DNA synthesis, a purine richsequence 5′ to the 3′ LTR that serve as the priming site for thesynthesis of the second strand of DNA synthesis, and specific sequencesnear the ends of the LTRs that enable the insertion of the DNA state ofthe retrovirus to insert into the host genome. The removal of the gag,pol, and env genes allows for about 8 kb of foreign sequence to beinserted into the viral genome, become reverse transcribed, and uponreplication be packaged into a new retroviral particle. This amount ofnucleic acid is sufficient for the delivery of a one to many genesdepending on the size of each transcript. It is preferable to includeeither positive or negative selectable markers along with other genes inthe insert.

Since the replication machinery and packaging proteins in mostretroviral vectors have been removed (gag, pol, and env), the vectorsare typically generated by placing them into a packaging cell line. Apackaging cell line is a cell line which has been transfected ortransformed with a retrovirus that contains the replication andpackaging machinery, but lacks any packaging signal. When the vectorcarrying the DNA of choice is transfected into these cell lines, thevector containing the gene of interest is replicated and packaged intonew retroviral particles, by the machinery provided in cis by the helpercell. The genomes for the machinery are not packaged because they lackthe necessary signals.

(2) Adenoviral Vectors

The construction of replication-defective adenoviruses has beendescribed (Berkner et al., J. Virology 61:1213-1220 (1987); Massie etal., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987);Zhang “Generation and identification of recombinant adenovirus byliposome-mediated transfection and PCR analysis” BioTechniques15:868-872 (1993)). The benefit of the use of these viruses as vectorsis that they are limited in the extent to which they can spread to othercell types, since they can replicate within an initial infected cell,but are unable to form new infectious viral particles. Recombinantadenoviruses have been shown to achieve high efficiency gene transferafter direct, in vivo delivery to airway epithelium, hepatocytes,vascular endothelium, CNS parenchyma and a number of other tissue sites(Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin.Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092(1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992);Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout,Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993);Caillaud, Eur. J. Neuroscience 5:1287-1291(1993); and Ragot, J. Gen.Virology 74:501-507 (1993)). Recombinant adenoviruses achieve genetransduction by binding to specific cell surface receptors, after whichthe virus is internalized by receptor-mediated endocytosis, in the samemanner as wild type or replication-defective adenovirus (Chardonnet andDales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985);Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., Mol. Cell.Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991);Wickham et al., Cell 73:309-319 (1993)).

A viral vector can be one based on an adenovirus which has had the E1gene removed and these virons are generated in a cell line such as thehuman 293 cell line. In another preferred embodiment both the E1 and E3genes are removed from the adenovirus genome.

(3) Adeno-Associated Viral Vectors

Another type of viral vector is based on an adeno-associated virus(AAV). This defective parvovirus is a preferred vector because it caninfect many cell types and is nonpathogenic to humans. AAV type vectorscan transport about 4 to 5 kb and wild type AAV is known to stablyinsert into chromosome 19. Vectors which contain this site specificintegration property are preferred. An especially preferred embodimentof this type of vector is the P4.1 C vector produced by Avigen, SanFrancisco, Calif., which can contain the herpes simplex virus thymidinekinase gene, HSV-tk, and/or a marker gene, such as the gene encoding thegreen fluorescent protein, GFP.

In another type of AAV virus, the AAV contains a pair of invertedterminal repeats (ITRs) which flank at least one cassette containing apromoter which directs cell-specific expression operably linked to aheterologous gene. Heterologous in this context refers to any nucleotidesequence or gene which is not native to the AAV or B19 parvovirus.

Typically the AAV and B19 coding regions have been deleted, resulting ina safe, noncytotoxic vector. The AAV ITRs, or modifications thereof,confer infectivity and site-specific integration, but not cytotoxicity,and the promoter directs cell-specific expression. U.S. Pat. No.6,261,834 is herein incorporated by reference for material related tothe AAV vector.

The disclosed vectors thus provide DNA molecules, which are capable ofintegration into a mammalian chromosome without substantial toxicity.

The inserted genes in viral and retroviral usually contain promoters,and/or enhancers to help control the expression of the desired geneproduct. A promoter is generally a sequence or sequences of DNA thatfunction when in a relatively fixed location in regard to thetranscription start site. A promoter contains core elements required forbasic interaction of RNA polymerase and transcription factors, and maycontain upstream elements and response elements.

(4) Large Payload Viral Vectors

Molecular genetic experiments with large human herpesviruses haveprovided a means whereby large heterologous DNA fragments can be cloned,propagated and established in cells permissive for infection withherpesviruses (Sun et al., Nature genetics 8: 33-41, 1994; Cotter andRobertson,. Curr Opin Mol Ther 5: 633-644, 1999). These large DNAviruses (herpes simplex virus (HSV) and Epstein-Barr virus (EBV), havethe potential to deliver fragments of human heterologous DNA>150 kb tospecific cells. EBV recombinants can maintain large pieces of DNA in theinfected B-cells as episomal DNA. Individual clones carried humangenomic inserts up to 330 kb appeared genetically stable The maintenanceof these episomes requires a specific EBV nuclear protein, EBNA1,constitutively expressed during infection with EBV. Additionally, thesevectors can be used for transfection, where large amounts of protein canbe generated transiently in vitro. Herpesvirus amplicon systems are alsobeing used to package pieces of DNA>220 kb and to infect cells that canstably maintain DNA as episomes.

Other useful systems include, for example, replicating andhost-restricted non-replicating vaccinia virus vectors.

b) Non-Nucleic Acid Based Systems

The disclosed compositions can be delivered to the target cells in avariety of ways. Those of skill in the art understand how to use thesedeliver systems both for nucleic acid and for non-nucleic acidcompositions. For example, the compositions can be delivered throughelectroporation, or through lipofection, or through calcium phosphateprecipitation. The delivery mechanism chosen will depend in part on thetype of cell targeted and whether the delivery is occurring for examplein vivo or in vitro.

Thus, the compositions can comprise, in addition to the disclosedpeptides or vectors for example, lipids such as liposomes, such ascationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionicliposomes. Liposomes can further comprise proteins to facilitatetargeting a particular cell, if desired. Administration of a compositioncomprising a compound and a cationic liposome can be administered to theblood afferent to a target organ or inhaled into the respiratory tractto target cells of the respiratory tract. Regarding liposomes, see,e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989);Felgner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat.No. 4,897,355. Furthermore, the compound can be administered as acomponent of a microcapsule that can be targeted to specific cell types,such as macrophages, or where the diffusion of the compound or deliveryof the compound from the microcapsule is designed for a specific rate ordosage.

In the methods described above which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), delivery of the compositions to cells canbe via a variety of mechanisms. As one example, delivery can be via aliposome, using commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art. In addition, the disclosednucleic acid or vector can be delivered in vivo by electroporation, thetechnology for which is available from Genetronics, Inc. (San Diego,Calif.) as well as by means of a SONOPORATION machine (ImaRxPharmaceutical Corp., Tucson, Ariz.).

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem.) 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Inmunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). These techniques can be used for avariety of other specific cell types. Vehicles such as “stealth” andother antibody conjugated liposomes (including lipid mediated drugtargeting to colonic carcinoma), receptor mediated targeting of DNAthrough cell specific ligands, lymphocyte directed tumor targeting, andhighly specific therapeutic retroviral targeting of murine glioma cellsin vivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

Nucleic acids that are delivered to cells which are to be integratedinto the host cell genome, typically contain integration sequences.These sequences are often viral related sequences, particularly whenviral based systems are used. These viral integration systems can alsobe incorporated into nucleic acids which are to be delivered using anon-nucleic acid based system of deliver, such as a liposome, so thatthe nucleic acid contained in the delivery system can be come integratedinto the host genome.

Other general techniques for integration into the host genome include,for example, systems designed to promote homologous recombination withthe host genome. These systems typically rely on sequence flanking thenucleic acid to be expressed that has enough homology with a targetsequence within the host cell genome that recombination between thevector nucleic acid and the target nucleic acid takes place, causing thedelivered nucleic acid to be integrated into the host genome. Thesesystems and the methods necessary to promote homologous recombinationare known to those of skill in the art.

c) In Vivo/Ex Vivo

As described above, the compositions can be administered in apharmaceutically acceptable carrier and can be delivered to the subjectscells in vivo and/or ex vivo by a variety of mechanisms well known inthe art (e.g., uptake of naked DNA, liposome fusion, intramuscularinjection of DNA via a gene gun, endocytosis and the like).

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The compositions can be introduced into the cells via anygene transfer mechanism, such as, for example, calcium phosphatemediated gene delivery, electroporation, microinjection orproteoliposomes. The transduced cells can then be infused (e.g., in apharmaceutically acceptable carrier) or homotopically transplanted backinto the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject.

d) Bone Specific Delivery Systems

It is preferred that the disclosed compositions are either specificallydelivered to bone cells, such as osteoblasts or osteoblasts or that thedelivered compositions are specifically expressed or activated in bonecells, such as osteoblasts.

Specific delivery can occur, by for example, isolation of the targetcell, such as an osteoblast, with delivery of the composition to theisolated osteoblast.

7. Expression Systems

The nucleic acids that are delivered to cells typically containexpression controlling systems. For example, the inserted genes in viraland retroviral systems usually contain promoters, and/or enhancers tohelp control the expression of the desired gene product. A promoter isgenerally a sequence or sequences of DNA that function when in arelatively fixed location in regard to the transcription start site. Apromoter contains core elements required for basic interaction of RNApolymerase and transcription factors, and may contain upstream elementsand response elements.

a) Viral Promoters and Enhancers

Preferred promoters controlling transcription from vectors in mammalianhost cells may be obtained from various sources, for example, thegenomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g. beta actin promoter. Theearly and late promoters of the SV40 virus are conveniently obtained asan SV40 restriction fragment which also contains the SV40 viral originof replication (Tiers et al., Nature) 273: 113 (1978)). The immediateearly promoter of the human cytomegalovirus is conveniently obtained asa HindIII E restriction fragment (Greenway, P. J. et al., Gene 18:355-360 (1982)). Of course, promoters from the host cell or relatedspecies also are useful herein.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′(Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3′(Lusky, M. L., et al., Mol. Cell Bio. 3: 1108 (1983)) to thetranscription unit. Furthermore, enhancers can be within an intron(Banerji, J. L. et al., Cell 33: 729 (1983)) as well as within thecoding sequence itself (Osborne, T. F., et al., Mol. Cell Bio. 4: 1293(1984)). They are usually between 10 and 300 bp in length, and theyfunction in cis. Enhancers f unction to increase transcription fromnearby promoters. Enhancers also often contain response elements thatmediate the regulation of transcription. Promoters can also containresponse elements that mediate the regulation of transcription.Enhancers often determine the regulation of expression of a gene. Whilemany enhancer sequences are now known from mammalian genes (globin,elastase, albumin, -fetoprotein and insulin), typically one will use anenhancer from a eukaryotic cell virus for general expression. Preferredexamples are the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

The promotor and/or enhancer may be specifically activated either bylight or specific chemical events which trigger their function. Systemscan be regulated by reagents such as tetracycline and dexamethasone.There are also ways to enhance viral vector gene expression by exposureto irradiation, such as gamma irradiation, or alkylating chemotherapydrugs.

In certain embodiments the promoter and/or enhancer region can act as aconstitutive promoter and/or enhancer to maximize expression of theregion of the transcription unit to be transcribed. In certainconstructs the promoter and/or enhancer region be active in alleukaryotic cell types, even if it is only expressed in a particular typeof cell at a particular time. A preferred promoter of this type is theCMV promoter (650 bases). Other preferred promoters are SV40 promoters,cytomegalovimus (full length promoter), and retroviral vector LTF.

It has been shown that all specific regulatory elements can be clonedand used to construct expression vectors that are selectively expressedin specific cell types such as melanoma cells. The glial fibrillaryacetic protein (GFAP) promoter has been used to selectively expressgenes in cells of glial origin.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contain a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs. In certaintranscription units, the polyadenylation region is derived from the SV40early polyadenylation signal and consists of about 400 bases. It is alsopreferred that the transcribed units contain other standard sequencesalone or in combination with the above sequences improve expressionfrom, or stability of, the construct.

b) Markers

The viral vectors can include nucleic acid sequence encoding a markerproduct. This marker product is used to determine if the gene has beendelivered to the cell and once delivered is being expressed. Preferredmarker genes are the E. Coli lacZ gene, which encodes β-galactosidase,and green fluorescent protein.

In some embodiments the marker may be a selectable marker. Examples ofsuitable selectable markers for mammalian cells are dihydrofolatereductase (DHFR), thymidine kinase, neomycin, neomycin analog G418,hydromycin, and puramycin. When such selectable markers are successfullytransferred into a mammalian host cell, the transformed mammalian hostcell can survive if placed under selective pressure. There are twowidely used distinct categories of selective regimes. The first categoryis based on a cell's metabolism and the use of a mutant cell line whichlacks the ability to grow independent of a supplemented media. Twoexamples are: CHO DHFR-cells and mouse LTK-cells. These cells lack theability to grow without the addition of such nutrients as thymidine orhypoxanthine. Because these cells lack certain genes necessary for acomplete nucleotide synthesis pathway, they cannot survive unless themissing nucleotides are provided in a supplemented media. An alternativeto supplementing the media is to introduce an intact DHFR or TK geneinto cells lacking the respective genes, thus altering their growthrequirements. Individual cells which were not transformed with the DHFRor TK gene will not be capable of survival in non-supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, (Southern P. and Berg,P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan,R. C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B.et al., Mol. Cell. Biol. 5: 410-−413 (1985)). The three examples employbacterial genes under eukaryotic control to convey resistance to theappropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid)or hygromycin, respectively. Others include the neomycin analog G418 andpuramycin.

c) Bone Specific Expression Systems

It is preferred that the disclosed compositions are either specificallydelivered to bone cells, such as osteoblasts or osteoblasts or that thedelivered compositions are specifically expressed or activated in bonecells, such as osteoblasts.

Specific expression can occur in bone cells, by using a bone specificpromoter such as type I collagen, alkaline phosphatase, osteonectin,osteocalcin, and the cbfa1 promoter.

8. Pharmaceutical Carriers/Delivery of Pharmaceutical Products

As described above, the compositions can also be administered in vivo ina pharmaceutically acceptable carrier. By “pharmaceutically acceptable”is meant a material that is not biologically or otherwise undesirable,i.e., the material may be administered to a subject, along with thenucleic acid or vector, without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art.

The compositions may be administered orally, parenterally (e.g.,intravenously), by intramuscular injection, by intraperitonealinjection, transdermally, extracorporeally, topically or the like,although topical intranasal administration or administration by inhalantis typically preferred. As used herein, “topical intranasaladministration” means delivery of the compositions into the nose andnasal passages through one or both of the nares and can comprisedelivery by a spraying mechanism or droplet mechanism, or throughaerosolization of the nucleic acid or vector. The latter may beeffective when a large number of animals is to be treatedsimultaneously. Administration of the compositions by inhalant can bethrough the nose or mouth via delivery by a spraying or dropletmechanism. Delivery can also be directly to any area of the respiratorysystem (e.g., lungs) via intubation. The exact amount of thecompositions required will vary from subject to subject, depending onthe species, age, weight and general condition of the subject, theseverity of the allergic disorder being treated, the particular nucleicacid or vector used, its mode of administration and the like. Thus, itis not possible to specify an exact amount for every composition.However, an appropriate amount can be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother, 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth” and otherantibody conjugated liposomes (including lipid mediated drug targetingto colonic carcinoma), receptor mediated targeting of DNA through cellspecific ligands, lymphocyte directed tumor targeting, and highlyspecific therapeutic retroviral targeting of murine glioma cells invivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

a) Pharmaceutically Acceptable Carriers

The compositions, including antibodies, can be used therapeutically incombination with a pharmaceutically acceptable carrier.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions may also includeone or more active ingredients such as antimicrobial agents,antiinflammatory agents, anesthetics, and the like.

The pharmaceutical composition may be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration may be topically (includingophthalmically, vaginally, rectally, intranasally), orally, byinhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection. The disclosedantibodies can be administered intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

b) Therapeutic Uses

The dosage ranges for the administration of the compositions are thoselarge enough to produce the desired effect in which the symptomsdisorder are effected. The dosage should not be so large as to causeadverse side effects, such as unwanted cross-reactions, anaphylacticreactions, and the like. Generally, the dosage will vary with the age,condition, sex and extent of the disease in the patient and can bedetermined by one of skill in the art. The dosage can be adjusted by theindividual physician in the event of any counterindications. Dosage canvary, and can be administered in one or more dose administrations daily,for one or several days.

Other compositions which do not have a specific pharmaceutical function,but which may be used for tracking changes within cellular chromosomesor for the delivery of diagnositc tools for example can be delivered inways similar to those described for the pharmaceutical products.

The compositions can also be used for example as tools to isolate andtest new drug candidates for a variety of diseases. They can also beused for the continued isolation and study, osteoblast/osteoclast lacunainteractions and functions. There use as exogenous DNA delivery devicescan be expanded for nearly any reason desired by those of skill in theart.

9. Chips and Micro Arrays

Disclosed are chips where at least one address is the sequences or partof the sequences set forth in any of the nucleic acid sequencesdisclosed herein. Also disclosed are chips where at least one address isthe sequences or portion of sequences set forth in any of the peptidesequences disclosed herein.

Also disclosed are chips where at least one address is a variant of thesequences or part of the sequences set forth in any of the nucleic acidsequences disclosed herein. Also disclosed are chips where at least oneaddress is a variant of the sequences or portion of sequences set forthin any of the peptide sequences disclosed herein.

10. Computer Readable Mediums

It is understood that the disclosed nucleic acids and proteins can berepresented as a sequence consisting of the nucleotides of amino acids.There are a variety of ways to display these sequences, for example thenucleotide guanosine can be represented by G or g. Likewise the aminoacid valine can be represented by Val or V. Those of skill in the artunderstand how to display and express any nucleic acid or proteinsequence in any of the variety of ways that exist, each of which isconsidered herein disclosed. Specifically contemplated herein is thedisplay of these sequences on computer readable mediums, such as,commercially available floppy disks, tapes, chips, hard drives, compactdisks, and video disks, or other computer readable mediums. Alsodisclosed are the binary code representations of the disclosedsequences. Those of skill in the art understands what computer readablemediums. Thus, computer readable mediums on which the nucleic acids orprotein sequences are recorded, stored, or saved.

Disclosed are computer readable mediums comprising the sequences andinformation regarding the sequences set forth herein. Also disclosed arecomputer readable mediums comprising the sequences and informationregarding the sequences set forth herein wherein the sequences do notinclude SEQ ID Nos: 37, 38, 39, 40, 41, and 42.

11. Compositions Identified by Screening With DisclosedCompositions/Combinatorial Chemistry a) Combinatorial Chemistry

The disclosed compositions can be used as targets for any combinatorialtechnique to identify molecules or macromolecular molecules thatinteract with the disclosed compositions in a desired way. The nucleicacids, peptides, and related molecules disclosed herein can be used astargets in any molecular modeling program or approach. Also disclosedare the compositions that are identified through combinatorialtechniques or screening techniques in which the compositions disclosedin SEQ ID NOS:1-50, for example, or portions thereof, are used as thetarget in a combinatorial or screening protocol.

It is understood that when using the disclosed compositions incombinatorial techniques or screening methods, molecules, such asmacromolecular molecules, will be identified that have particulardesired properties such as inhibition or stimulation of the targetmolecule's function. The molecules identified and isolated when usingthe disclosed compositions, such as, GPC4 or TRAP or TRIP or SEQ ID NO:5or SEQ ID NO23, are also disclosed. Thus, the products produced usingthe combinatorial or screening approaches that involve the disclosedcompositions are also considered herein disclosed.

Combinatorial chemistry includes but is not limited to all methods forisolating small molecules or macromolecules that are capable of bindingeither a small molecule or another macromolecule, typically in aniterative process. Proteins, oligonucleotides, and sugars are examplesof macromolecules. For example, oligonucleotide molecules with a givenfunction, catalytic or ligand-binding, can be isolated from a complexmixture of random oligonucleotides in what has been referred to as “invitro genetics” (Szostak, TIBS 19:89, 1992). One synthesizes a largepool of molecules bearing random and defined sequences and subjects thatcomplex mixture, for example, approximately 10¹⁵ individual sequences in100 μg of a 100 nucleotide RNA, to some selection and enrichmentprocess. Through repeated cycles of affinity chromatography and PCRamplification of the molecules bound to the ligand on the column,Ellington and Szostak (1990) estimated that 1 in 10¹⁰ RNA moleculesfolded in such a way as to bind a small molecule dyes. DNA moleculeswith such ligand-binding behavior have been isolated as well (Ellingtonand Szostak, 1992; Bock et al, 1992). Techniques aimed at similar goalsexist for small organic molecules, proteins, antibodies and othermacromolecules known to those of skill in the art. Screening sets ofmolecules for a desired activity whether based on small organiclibraries, oligonucleotides, or antibodies is broadly referred to ascombinatorial chemistry. Combinatorial techniques are particularlysuited for defining binding interactions between molecules and forisolating molecules that have a specific binding activity, often calledaptamers when the macromolecules are nucleic acids.

There are a number of methods for isolating proteins which either havede novo activity or a modified activity. For example, phage displaylibraries have been used to isolate numerous peptides that interact witha specific target. (See for example, U.S. Pat. Nos. 6,031,071;5,824,520; 5,596,079; and 5,565,332 which are herein incorporated byreference at least for their material related to phage display andmethods relate to combinatorial chemistry)

A preferred method for isolating proteins that have a given function isdescribed by Roberts and Szostak (Roberts R. W. and Szostak J. W. Proc.Natl. Acad. Sci. USA, 94(23)12997-302 (1997). This combinatorialchemistry method couples the functional power of proteins and thegenetic power of nucleic acids. An RNA molecule is generated in which apuramycin molecule is covalently attached to the 3′-end of the RNAmolecule. An in vitro translation of this modified RNA molecule causesthe correct protein, encoded by the RNA to be translated. In addition,because of the attachment of the puramycin, a peptidyl acceptor whichcannot be extended, the growing peptide chain is attached to thepuramycin which is attached to the RNA. Thus, the protein molecule isattached to the genetic material that encodes it. Normal in vitroselection procedures can now be done to isolate functional peptides.Once the selection procedure for peptide function is completetraditional nucleic acid manipulation procedures are performed toamplify the nucleic acid that codes for the selected functionalpeptides. After amplification of the genetic material, new RNA istranscribed with puramycin at the 3′-end, new peptide is translated andanother functional round of selection is performed. Thus, proteinselection can be performed in an iterative manner just like nucleic acidselection techniques. The peptide which is translated is controlled bythe sequence of the RNA attached to the puramycin. This sequence can beanything from a random sequence engineered for optimum translation (i.e.no stop codons etc.) or it can be a degenerate sequence of a known RNAmolecule to look for improved or altered function of a known peptide.The conditions for nucleic acid amplification and in vitro translationare well known to those of ordinary skill in the art and are preferablyperformed as in Roberts and Szostak (Roberts R. W. and Szostalck J. W.Proc. Natl. Acad. Sci. USA, 94(23)12997-302 (1997)).

Another preferred method for combinatorial methods designed to isolatepeptides is described in Cohen et al. (Cohen B. A., et al., Proc. Natl.Acad. Sci. USA 95(24):14272-7 (1998)). This method utilizes and modifiestwo-hybrid technology. Yeast two-hybrid systems are useful for thedetection and analysis of protein:protein interactions. The two-hybridsystem, initially described in the yeast Saccharomyces cerevisiae, is apowerful molecular genetic technique for identifying new regulatorymolecules, specific to the protein of interest (Fields and Song, Nature340:245-6 (1989)). Cohen et al., modified this technology so that novelinteractions between synthetic or engineered peptide sequences could beidentified which bind a molecule of choice. The benefit of this type oftechnology is that the selection is done in an intracellularenvironment. The method utilizes a library of peptide molecules thatattached to an acidic activation domain. A peptide of choice, forexample an extracellular portion of GPC4 is attached to a DNA bindingdomain of a transcriptional activation protein, such as Gal 4. Byperforming the Two-hybrid technique on this type of system, moleculesthat bind the extracellular portion of GPC4 can be identified.

Using methodology well known to those of skill in the art, incombination with various combinatorial libraries, one can isolate andcharacterize those small molecules or macromolecules, which bind to orinteract with the desired target. The relative binding affinity of thesecompounds can be compared and optimum compounds identified usingcompetitive binding studies, which are well known to those of skill inthe art.

Techniques for making combinatorial libraries and screeningcombinatorial libraries to isolate molecules which bind a desired targetare well known to those of skill in the art. Representative techniquesand methods can be found in but are not limited to U.S. Pat. Nos.5,084,824, 5,288,514, 5,449,754, 5,506,337, 5,539,083, 5,545,568,5,556,762, 5,565,324, 5,565,332, 5,573,905, 5,618,825, 5,619,680,5,627,210, 5,646,285, 5,663,046, 5,670,326, 5,677,195, 5,683,899,5,688,696, 5,688,997, 5,698,685, 5,712,146, 5,721,099, 5,723,598,5,741,713, 5,792,431, 5,807,683, 5,807,754, 5,821,130, 5,831,014,5,834,195, 5,834,318, 5,834,588, 5,840,500, 5,847,150, 5,856,107,5,856,496, 5,859,190, 5,864,010, 5,874,443, 5,877,214, 5,880,972,5,886,126, 5,886,127, 5,891,737, 5,916,899, 5,919,955, 5,925,527,5,939,268, 5,942,387, 5,945,070, 5,948,696, 5,958,702, 5,958,792,5,962,337, 5,965,719, 5,972,719, 5,976,894, 5,980,704, 5,985,356,5,999,086, 6,001,579, 6,004,617, 6,008,321, 6,017,768, 6,025,371,6,030,917, 6,040,193, 6,045,671, 6,045,755, 6,060,596, and 6,061,636.

Combinatorial libraries can be made from a wide array of molecules usinga number of different synthetic techniques. For example, librariescontaining fused 2,4-pyrimidinediones (U.S. Pat. No. 6,025,371)dihydrobenzopyrans (U.S. Pat. Nos. 6,017,768and 5,821,130), amidealcohols (U.S. Pat. No. 5,976,894), hydroxy-amino acid amides (U.S. Pat.No. 5,972,719) carbohydrates (U.S. Pat. No. 5,965,719),1,4-benzodiazepin-2,5-diones (U.S. Pat. No. 5,962,337), cyclics (U.S.Pat. No. 5,958,792), biaryl amino acid amides (U.S. Pat. No. 5,948,696),thiophenes (U.S. Pat. No. 5,942,387), tricyclic Tetrahydroquinolines(U.S. Pat. No. 5,925,527), benzofurans (U.S. Pat. No. 5,919,955),isoquinolines (U.S. Pat. No. 5,916,899), hydantoin and thiohydantoin(U.S. Pat. No. 5,859,190), indoles (U.S. Pat. No. 5,856,496),imidazol-pyrido-indole and imidazol-pyrido-benzothiophenes (U.S. Pat.No. 5,856,107) substituted 2-methylene-2,3-dihydrothiazoles (U.S. Pat.No. 5,847,150), quinolines (U.S. Pat. No. 5,840,500), PNA (U.S. Pat. No.5,831,014), containing tags (U.S. Pat. No. 5,721,099), polypeptides(U.S. Pat. No. 5,712,146), morpholino-subunits (U.S. Pat. Nos. 5,698,685and 5,506,337), sulfamides (U.S. Pat. No. 5,618,825), andbenzodiazepines (U.S. Pat. No. 5,288,514).

Screening peptides similar to the peptide set forth in SEQ ID NO:23 forbinding to TRAP or osteoclast lacuna, for example, is a method ofisolating desired compounds.

Molecules isolated which bind TRAP can either be competitive inhibitorsor non-competitive inhibitors or osteoblast or GPC4 or TRIPinteractions. In certain embodiments the inhibitors of osteoblastbinding to osteoclast lacuna are non-competitive inhibitors and in otherembodiments the compositions can be competitive inhibitors. One type ofnon-competitive inhibitor will cause allosteric rearrangements whichprevent osteoblasts from binding to osteoclast lacuna.

As used herein combinatorial methods and libraries included traditionalscreening methods and libraries as well as methods and libraries used ininterative processes.

b) Computer Assisted Drug Design

The disclosed compositions can be used as targets for any molecularmodeling technique to identify either the structure of the disclosedcompositions or to identify potential or actual molecules, such as smallmolecules, which interact in a desired way with the disclosedcompositions. The nucleic acids, peptides, and related moleculesdisclosed herein can be used as targets in any molecular modelingprogram or approach.

It is understood that when using the disclosed compositions in modelingtechniques, molecules, such as macromolecular molecules, will beidentified that have particular desired properties such as inhibition orstimulation or the target molecule's function. The molecules identifiedand isolated when using the disclosed compositions, such as, SEQ ID NOs:1-50, are also disclosed. Thus, the products produced using themolecular modeling approaches that involve the disclosed compositions,such as, SEQ ID NOs: 1-50 are also considered herein disclosed.

Thus, one way to isolate molecules that bind a molecule of choice isthrough rational design. This is achieved through structural informationand computer modeling. Computer modeling technology allows visualizationof the three-dimensional atomic structure of a selected molecule and therational design of new compounds that will interact with the molecule.The three-dimensional construct typically depends on data from x-raycrystallographic analyses or NMR imaging of the selected molecule. Themolecular dynamics require force field data. The computer graphicssystems enable prediction of how a new compound will link to the targetmolecule and allow experimental manipulation of the structures of thecompound and target molecule to perfect binding specificity. Predictionof what the molecule-compound interaction will be when small changes aremade in one or both requires molecular mechanics software andcomputationally intensive computers, usually coupled with user-friendly,menu-driven interfaces between the molecular design program and theuser.

Examples of molecular modeling systems are the CHARMm and QUANTAprograms, Polygen Corporation, Waltham, Mass. CHARMm performs the energyminimization and molecular dynamics functions. QUANTA performs theconstruction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive withspecific proteins, such as Rotivinen, et al., 1988 Acta PharmaceuticaFennica 97, 159-166; Ripka, New Scientist 54-57 (Jun. 16, 1988);McKinaly and Rossmann, 1989 Annu. Rev. Pharmacol. _Toxiciol. 29,111-122; Perry and Davies, QSAR: Quantitative Structure-ActivityRelationships in Drug Design pp. 189-193 (Alan R. Liss, Inc. 1989);Lewis and Dean, 1989 Proc. R. Soc. Lond. 236, 125-140 and 141-162; and,with respect to a model enzyme for nucleic acid components, Askew, etal., 1989 J. Am. Chem. Soc. 111, 1082-1090. Other computer programs thatscreen and graphically depict chemicals are available from companiessuch as BioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga,Ontario, Canada, and Hypercube, Inc., Cambridge, Ontario. Although theseare primarily designed for application to drugs specific to particularproteins, they can be adapted to design of molecules specificallyinteracting with specific regions of DNA or RNA, once that region isidentified.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds whichalter substrate binding or enzymatic activity.

12. Kits

Disclosed herein are kits that are drawn to reagents that can be used inpracticing the methods disclosed herein. The kits can include anyreagent or combination of reagent discussed herein or that would beunderstood to be required or beneficial in the practice of the disclosedmethods. For example, the kits could include primers to perform theamplification reactions discussed in certain embodiments of the methods,as well as the buffers and enzymes required to use the primers asintended. For example, disclosed is a kit for assessing a subject's riskfor acquiring osteoporosis, comprising the probes or primers related tosequences set forth in SEQ ID Nos:38 (GPC4), 40(TRIP), and 42(TRAP).

13. Functional Equivalents

It is understood that the compositions disclosed herein have certainfunctions, such as binding TRAP or binding TRIP or GPC4 or osteoblastcells, osteoclast cells, or osteoclast lacuna. Disclosed herein arecertain structural requirements for performing the disclosed functions,and it is understood that there are a variety of structures which canperform the same function which are related to the disclosed structures,and that these structures will ultimately achieve the same result, forexample stimulation or inhibition of osteoblast binding to osteoclastlacuna.

D. Methods 1. Methods of Making the Compositions

The compositions disclosed herein and the compositions necessary toperform the disclosed methods can be made using any method known tothose of skill in the art for that particular reagent or compound unlessotherwise specifically noted. Methods for protein synthesis orproduction

a) Nucleic Acid Synthesis

For example, the nucleic acids, such as, the oligonucleotides to be usedas primers can be made using standard chemical synthesis methods or canbe produced using enzymatic methods or any other known method. Suchmethods can range from standard enzymatic digestion followed bynucleotide fragment isolation (see for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) topurely synthetic methods, for example, by the cyanoethyl phosphoramiditemethod using a Milligen or Beckman System 1Plus DNA synthesizer (forexample, Model 8700 automated synthesizer of Milligen-Biosearch,Burlington, Mass. or ABI Model 380B). Synthetic methods useful formaking oligonucleotides are also described by Ikuta et al., Ann. Rev.Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triestermethods), and Narang et al., Methods Enzymol., 65:610-620 (1980),(phosphotriester method). Protein nucleic acid molecules can be madeusing known methods such as those described by Nielsen et al.,Bioconjug. Chem. 5:3-7 (1994).

Nucleic acids can also be produced and replicated and manufactured usingany known recombinant molecular biology protocol. These methods can befound for example in Sambrook et al., Molecular Cloning: A LaboratoryManual, 3rd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 2001 which is herein incorporated by reference specifically atleast for material related to the manipulation, such as production, ofbiological macromolecules such as nucleic acids and proteins.

b) Peptide Synthesis

One method of producing the disclosed proteins, such as SEQ ID NO:23, isto link two or more peptides or polypeptides together by proteinchemistry techniques. For example, peptides or polypeptides can bechemically synthesized using currently available laboratory equipmentusing either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc(tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., FosterCity, Calif.). One skilled in the art can readily appreciate that apeptide or polypeptide corresponding to the disclosed proteins, forexample, can be synthesized by standard chemical reactions. For example,a peptide or polypeptide can be synthesized and not cleaved from itssynthesis resin whereas the other fragment of a peptide or protein canbe synthesized and subsequently cleaved from the resin, thereby exposinga terminal group which is functionally blocked on the other fragment. Bypeptide condensation reactions, these two fragments can be covalentlyjoined via a peptide bond at their carboxyl and amino termini,respectively, to form an antibody, or fragment thereof. (Grant G A(1992) Synthetic Peptides: A User Guide. W.H. Freeman and Co., N.Y.(1992); Bodansky M and Trost B., Ed. (1993) Principles of PeptideSynthesis. Springer-Verlag Inc., NY (which is herein incorporated byreference at least for material related to peptide synthesis).Alternatively, the peptide or polypeptide is independently synthesizedin vivo as described herein. Once isolated, these independent peptidesor polypeptides may be linked to form a peptide or fragment thereof viasimilar peptide condensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentsallow relatively short peptide fragments to be joined to produce largerpeptide fragments, polypeptides or whole protein domains (Abrahmsen L etal., Biochemistry, 30:4151 (1991)). Alternatively, native chemicalligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method consists of a two step chemical reaction (Dawson et al.Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779(1994)). The first step is the chemoselective reaction of an unprotectedsynthetic peptide—thioester with another unprotected peptide segmentcontaining an amino-terminal Cys residue to give a thioester-linkedintermediate as the initial covalent product. Without a change in thereaction conditions, this intermediate undergoes spontaneous, rapidintramolecular reaction to form a native peptide bond at the ligationsite (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I etal., J. Biol. Chem., 269:16075 (1994); Clark-Lewis I et al.,Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry33:6623-30 (1994)).

Alternatively, unprotected peptide segments are chemically linked wherethe bond formed between the peptide segments as a result of the chemicalligation is an unnatural (non-peptide) bond (Schnolzer, M et al.Science, 256:221 (1992)). This technique has been used to synthesizeanalogs of protein domains as well as large amounts of relatively pureproteins with full biological activity (deLisle Milton R C et al.,Techniques in Protein Chemistry IV. Academic Press, New York, pp.257-267 (1992)).

c) Processes for Making the Compositions

Disclosed are processes for making the compositions as well as makingthe intermediates leading to the compositions. For example, disclosedare nucleic acids in SEQ ID NOs:1-19, 37, 39, 41. There are a variety ofmethods that can be used for making these compositions, such assynthetic chemical methods and standard molecular biology methods. It isunderstood that the methods of making these and the other disclosedcompositions are specifically disclosed.

Disclosed are nucleic acid molecules produced by the process comprisinglinking in an operative way a nucleic acid comprising the sequence setforth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:37, SEQ ID NO: 39, orSEQ ID NO:41 and a sequence controlling the expression of the nucleicacid.

Also disclosed are nucleic acid molecules produced by the processcomprising linking in an operative way a nucleic acid moleculecomprising a sequence having 80% identity to a sequence set forth in SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:37, SEQ ID NO: 39, or SEQID NO:41, and a sequence controlling the expression of the nucleic acid.

Disclosed are nucleic acid molecules produced by the process comprisinglinking in an operative way a nucleic acid molecule comprising asequence that hybridizes under stringent hybridization conditions to asequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:37,SEQ ID NO: 39, or SEQ ID NO:41 and a sequence controlling the expressionof the nucleic acid.

Disclosed are nucleic acid molecules produced by the process comprisinglinking in an operative way a nucleic acid molecule comprising asequence encoding a peptide set forth in SEQ ID NO:19, SEQ ID NO:20, SEQID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO: 40, or SEQ ID NO:42 and a sequencecontrolling an expression of the nucleic acid molecule.

Disclosed are nucleic acid molecules produced by the process comprisinglinking in an operative way a nucleic acid molecule comprising asequence encoding a peptide having 80% identity to a peptide set forthin SEQ ID NO:19, SEQ ED NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO: 40,or SEQ ID NO:42 and a sequence controlling an expression of the nucleicacid molecule.

Disclosed are nucleic acids produced by the process comprising linkingin an operative way a nucleic acid molecule comprising a sequenceencoding a peptide having 80% identity to apeptide set forth in SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:18, SEQ ID NO:38, SEQ ID NO: 40, or SEQID NO:42, wherein any change from the SEQ ID Nos:19-36, SEQ ID NO:37,SEQ ID NO: 39, and SEQ ID NO:41 are conservative changes and a sequencecontrolling an expression of the nucleic acid molecule.

Disclosed are cells produced by the process of transforming the cellwith any of the disclosed nucleic acids. Disclosed are cells produced bythe process of transforming the cell with any of the non-naturallyoccurring disclosed nucleic acids.

Disclosed are any of the disclosed peptides produced by the process ofexpressing any of the disclosed nucleic acids. Disclosed are any of thenon-naturally occurring disclosed peptides produced by the process ofexpressing any of the disclosed nucleic acids. Disclosed are any of thedisclosed peptides produced by the process of expressing any of thenon-naturally disclosed nucleic acids.

Disclosed are animals produced by the process of transfecting a cellwithin the animal with any of the nucleic acid molecules disclosedherein. Disclosed are animals produced by the process of transfecting acell within the animal any of the nucleic acid molecules disclosedherein, wherein the animal is a mammal. Also disclosed are animalsproduced by the process of transfecting a cell within the animal any ofthe nucleic acid molecules disclosed herein, wherein the mammal ismouse, rat, rabbit, cow, sheep, pig, or primate.

Also disclose are animals produced by the process of adding to theanimal any of the cells disclosed herein.

2. Methods of Using the Compositions

Disclosed are methods of regulating bone formation which compriseadministering to a patient in need of such regulation a peptidecomprising an amino acid sequence set forth in SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40 for a time and underconditions sufficient to regulate bone formation.

Also disclosed are methods of regulating bone formation which compriseadministering to a patient in need of such regulation a peptidecomprising an amino acid sequence having at least 80% identity to thepeptides set forth in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ED NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:38, or SEQ ID NO:40 for a time and under conditions sufficient toregulate bone formation.

Disclosed are methods, wherein the peptide is administered by systemicmeans or wherein the systemic means is intravenous or intra-arterialinfusion or wherein the peptide is administered by implanting on bone.

Disclosed are methods of stimulating bone formation in a bone cellculture which comprise adding a peptide having an amino acid sequenceset forth in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, or SEQ IDNO:40 to a bone cell culture.

Disclosed are methods, wherein the bone cell culture comprisesosteoblast cells or wherein the bone cell culture comprises osteoclastcells.

Disclosed are methods of regulating bone formation which compriseadministering to a patient in need of such regulation a nucleotidemolecule comprising a sequence set forth in SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:37, or SEQ ID NO:39 or the peptide produced from thesenucleic acid molecules for a time and under conditions sufficient toregulate bone formation.

Disclosed are methods of regulating bone formation which compriseadministering to a patient in need of such regulation a nucleotidemolecule comprising a sequence set forth in SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:37, or SEQ ID NO:39 for a time and under conditionssufficient to regulate bone formation.

Disclosed are methods, wherein the nucleotide molecule transfects a bonecell or wherein the bone cell is an osteoblast cell. Methods to deliverspecific proteins would include viruses that would infect all cells inthe area and produce the protein of interest. Constructs that would bebone specific can be utilized, as well as ex vivo transfection in thelaboratory and return of the patients cells to the site of interest,i.e. a fracture site or implant site. Also, manipulation of embryonicstem cells to produce the protein of interest and then differentiate itinto osteoblasts can be performed.

Disclosed are methods of regulating bone formation which compriseadministering to a patient in need of such regulation a cell which hasbeen transformed with a nucleotide molecule having a sequence set forthin SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:37, or SEQ ID NO:39 for atime and under conditions sufficient to regulate bone formation.

Disclosed are methods, wherein the cell is a fibroblast cell, acartilage cell, a bone marrow cell, a stem cell, or an adipocyte cell orwherein the nucleotide sequence is under the control of a promoter whichfunctions in the cells.

Disclosed are methods of stimulating bone formation in a bone cellculture which comprise adding any one of the cells disclosed herein tothe bone cell culture.

Disclosed are methods of promoting bone growth comprising contacting anarea where bone growth is desired with a composition comprising tartrateresistant acid phosphatase

Also disclosed are methods, wherein the composition further comprises animplant or methods wherein the implant is a dental implant.

Disclosed are methods of regulating bone growth comprising regulatingthe expression of GPC4 or TRIP in osteoblast cells.

Disclosed are methods, wherein the expression of GPC4 or TRIP isincreased or methods wherein the expression of GPC4 or TRIP is decreasedor methods wherein the expression of GPC4 or TRIP is regulated by anantisense molecule hybridizing to the mRNA of GPC4 or TRIP.

Disclosed are methods of producing an animal comprising introducing intothe animal a cell containing a nucleic acid molecule set forth in SEQ IDNOS:1-18, 37, 39, 41, or any variants herein disclosed, such that thenucleic acid will be expressed in the animal.

Creation of a transgenic animal that would produce large amounts of aspecific protein under the control of switchable promoter is disclosed.This has been done with the “tet”promoter. Genetic constructs placeddown stream of a tetracycline inducible promoter can be “turned on” inan animal at any time by administering tetracycline. Knock-out of agiven protein can be achieved by replacing the natural gene with a genethat has the initiation site ablated. No functional protein is thensynthesized. Disclosed are the knockins and knockouts of the disclosednucleic acids disclosed herein, including SEQ ID NOS:1-18, 37, 39, and41. Also disclosed are animals that are expressing the nucleic acids orpeptides disclosed herein.

Over-expression of either GFC4 or TRIP in osteoblasts (or other celltypes) would allow control of the site and amount of bone formation.Delivery of these agents by gene therapeutic techniques or pharmacologicagents that upregulate GPC4 and TRIP would be two ways to induce cell toproduce the molecules. Moreover, identification of the substrates thatbind to GPC4 and TRIP can provide a synthetic stimulatory environmentfor the formation of new bone. This could be used around orthopaedic anddental implants to more securely anchor the prostheses in bone.

Another use for these molecules may be as a diagnostic tool. Assays forthe level of either or both of GPC4 and TRIP can be used to predictmetabolic bone diseases such as osteoporosis and/or osteopetrosis.

a) Methods of Using the Compositions as Research and Diagnostic Tools

The disclosed compositions can be used in a variety of ways as researchtools. For example, the disclosed compositions, such as SEQ ID NOs:19-36can be used to study the interactions between osteoblasts and osteoclastlacuna, by for example acting as inhibitors of binding.

The compositions can be used for example as targets in combinatorialchemistry protocols or other screening protocols to isolate moleculesthat possess desired functional properties related to osteoblasts andosteoclasts.

The disclosed compositions can also be used diagnostic tools related todiseases of the bone, such as osteoporosis or other bone maladies. Apartial list of bone maladies is shown in Table 3. TABLE 3 type Ipostmenopausal sarcoidosis osteoporosis type II age-related osteoporosisdiabetes male osteoporosis osteomalacia secondary osteoporosis due toVDRR, VDDR, and nutritional rickets steroid or pharmaceutical usehypervitaminosis A and D renal osteodystrophy Paget's Disease renalstones osteopetrosis juvenile idiopathic osteoporosis skeletal tumorshyperparathyroidism rheumatoid and osteo arthritis hyperthyroidismosteogensis imperfecta hypercalcemia's chondrodystrophies Fanconisyndrome sclerosing bone dysplasias

The disclosed compositions can be used as discussed herein as eitherreagents in micro arrays or as reagents to probe or analyze existingmicroarrays. The disclosed compositions can be used in any known methodfor isolating or identifying single nucleotide polymorphisms. Thecompositions can also be used in any method for determining allelicanalysis of for example, GPC4, TRIP, or TRAP, particularly allelicanalysis as it relates to osteoblast and osteoclast lacuna interactionsand functions. The compositions can also be used in any known method ofscreening assays, related to chip/micro arrays. The compositions canalso be used in any known way of using the computer readable embodimentsof the disclosed compositions, for example, to study relatedness or toperform molecular modeling analysis related to the disclosedcompositions.

b) Pharmaceutical Methods

The disclosed compositions can in certain embodiments be delivered aspharmaceutical reagents, based on their ability to inhibit binding ofosteoblasts to osteoclasts. Many bone disease states are related toinappropriate osteoblast/osteoclast interactions. For example,compositions that bind TRAP and inhibit osteoblast binding to osteoclastlacuna can be used for example to modulate processes such as heterotopicbone formation, osteophyte formation, diffuse idiopathic skeletalhyperostosis (DISH), and myositis ossificans progressiva (MOP), etc.

E. EXAMPLES

Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.), but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. or is at ambient temperature, and pressure is ator near atmospheric.

1. Example 1 Phage Display and Isolation of Molecules That Bind TRAP a)Osteoblasts Bind and Differentiate in Resorption Lacunae

Resorption lacunae formed on the surface of cortical bone wafers (by amodification of the methods published by Dempster and Chambers, 33-35)were used as substrate surfaces on which to study osteoblast behavior.By all criteria, these lacunae represent the result of authenticosteoclast activity. Moreover, they resemble in vivo lacunae by virtueof their surface characteristics (SEM) and lysosomal content(immunocytochemistry). For example, it was shown that: i) that there isa high concentration of mannosylated carbohydrate side chains onresident lysosomal enzymes in lacunae, ii) that TRAP is prevalent alongresorbed surfaces and iii) that osteoblasts can adhere within thelacunae. (Data showed FITC conjugated lectin (Pisum sativum) withspecificity for mannose carbohydrate structures binds only withinosteoclast lacunae (dotted lines) on cortical bone wafers. Data alsoshowed that FITC conjugated lectin (concanavalin A) with specificity formannose and glucose structures binds only within osteoclast lacunae(dotted lines) on cortical bone wafers. In addition data showed crosssections of an osteoclast pit in a cortical bone wafer demonstrated thatthe presence of TRAP in the pit area by immunocytochemistry and dataalso showed an osteoblast binding within a resorption lacunae on acortical bone wafer.)

Bone wafers have been used to demonstrate that osteoblasts bind withmuch higher affinity to resorption surfaces than unresorbed surfaces andthat when they bind they alter their phenotype toward a moredifferentiated state.

FIG. 3 a shows that there is a positive correlation between the extentof osteoclast pitting on a wafer and the affinity of the osteoblasts tobind. FIG. 3 b demonstrates that osteoblasts residing on pitted wafersproduce more alkaline phosphatase per cell and proliferate at a greaterrate. Data also showed a visual demonstration of alkaline phosphataseactivity only in osteoblasts residing in resorption pits. Data showedthat osteoblast cultured on pitted bone wafers, and that only cellsvisibly producing alkaline phosphatase (staining) reside withinosteoclast lacunae. In control experiments the wafers have been treatedwith glycosidases and boiling and have been able to eliminate thebinding and differentiation effects. Thus, these data document thatosteoblasts can use specific site-directing signals for attachment anddifferentiation and that the signals can be removed with enzymatictreatments and boiling.

Exposing osteoblasts in culture directly to soluble lysosomal enzymesvalidated these results. These data have been published and documentthat soluble TRAP has many of the differentiation and proliferationcharacteristics of TRAP bound to resorption surfaces. A compilation ofthese data is presented in FIG. 4.

These results show that 1) Osteoblasts bind and differentiate inresorption lacunae, 2) Removal of soluble factors from the lacunaeeliminates the effect, and 3) Soluble TRAP can mimic some of thedifferentiative and proliferative effects of osteoblasts binding tolacunae.

b) Phage Display

In phage display either random peptide libraries or cell specific cDNAlibraries can be used. Both have been used to target the interactionsthat take place in bone resporbtion and formation. Generally, thepeptides or proteins are expressed on the surface coat of M13 or T7phage and then these phage are used in a biopanning technique toidentify which phage have affinity for a particular substrate (the“bait”). The adherent phage are eluted, amplified and used in anotherround of panning. After at least three rounds, phage clones aresequenced. This process is diagrammatically shown in FIG. 2.

(1) Random Phage Library

The type V tartrate resistant acid phosphatase (TRAP) was used as baitwith a random peptide phage display library. TRAP was selected becauseof its association with osteoclast bone resorptive activity and becauseit has been demonstrated that it remains bound to lacunae (Chambers T J.et al., ournal of Cellular Physiology. 132(1):90-6, 1987 July 87250990;Murrills R J. et al., Endocrinology. 127(6):2648-53, 1990; Wergedal J E.Baylink D J., Journal of Histochemnistry & Cytochemistry.17(12):799-806, 1969; Murrills R J. et al., Journal of Bone & MineralResearch. 4(2):259-68, 1989). The TRAP used in these experiments waspurified to homogeneity and was generously provided by Dr. M. Roberts.

A random 12 amino acid oligopeptide expression library in M13 phage wasused to probe TRAP that was immobilized on a tissue culture dish. Afterthree rounds of bio-panning, elution and amplification 44 phage cloneswere selected at random and sequenced. Nine different sequences wereidentified. They are listed in Table 4 along with their consensusfrequency. The most frequent phage sequenced was represented 15 timesand the least frequent only a single time. TABLE 4 Results of PhageDisplay Bio-Panning with TRAP SEQ, ID No. Table 1: DNA and PeptideSequence Fre SEQ. ID No. I CACTCTACTATGGGTTTTACGGCTCCGCCGCATTAT  1144SEQ. ID No. 19             HSTMGFrAPPHY SEQ. ID No. 2ACTATGGGTTTTACGGCTCCGCGGTTTCCGCATTAT 6/44 SEQ. ID No. 20            TMGFrAPRFPHY SEQ. ID No. 3TCTCAGTGGCATCCGCGGTCTGCGTCGTATCCGATG 4/44 SEQ. ID No. 21            SQWHPRSASYPM SEQ. ID No. 4ACGCCGTCTCTTCCTCCGACTATGTTTCGGTTGACT 4/44 SEQ. ID No. 22            TPSLPPTMFRLT SEQ. ID No. 5ACGCCGCTTTCGTATCTGAAGGGTCTGGTGACGGTG 15144 SEQ. ID No. 23            TPLSYLKGLVTV SEQ. ID No. 6CGGCCACGGAACACCAGTAGACGTCCCATGCGCAGA 2/44 SEQ. ID No. 24            SAHGTSTGVPWP SEQ. ID No. 7ATAATGCGGAAACCGCGGAGCCGTAAAACCCATAGT 5/44 SEQ. ID No. 25            TMGFrAPRFPHY SEQ. ID No. 8CGGCCAACGGAACACCAGTAGACGTCCCATGCGCAG 2/44 SEQ. ID No. 26            LRMGRLLVFRWP SEQ. ID No. 9ACCAAGCCCCGAATCACGCGAATAAAGCGGCCAAGA 2/44 SEQ. ID No. 27            SWPLYSRDSGLG SEQ. ID No. 10CAGAGCCTCCTTCGTCCAATTTCACACACTAAGCCC n.d. SEQ. ID No. 28            GLSVEIGRRRL SEQ. ID No. 11CCGATCATACATAACCGAAATCAGATGAAACGGCAG n.d. SEQ. ID No. 29            LPFHLISVMYDR SEQ. ID No. 12CATCTTGCGCCGATGCCTCGGGCGTTGCATACGGGT n.d. SEQ. ID No. 30            HLAPMPRALHTG SEQ. ID No. 13GGGCTTAGTGTGTGAAATTGGACGAAGGAGGCTCTG n.d. SEQ. ID No. 31            GLSV-NWTKEAL SEQ. ID No. 14CATCTTGCGCCGATGCCTCGGGCGTTGCATACGGGT n.d. SEQ. ID No. 32            HLAPMPRALHTG SEQ. ID No. 15TTTGTGAAGCCTAAGGCGCTGTCTCTGCAGGCTGTG n.d. SEQ. ID No. 33            FVKPKALSLQAV SEQ. ID No. 16TTTCATGTGAATCCGACGTCTCCGACGCATCCGTTG n.d. SEQ. ID No. 34            FHVNPTSPTHPL SEQ. ID No. 17CAGCATGCGAATCATCAGGCTTGGAATAATCTTCGT n.d. SEQ. ID No. 35            QHANHQAWNNLR SEQ. ID No. 18TTTCATGTGAATCCGACGTCTCCGACGCATCCGTTG n.d. SEQ. ID No. 36            FHVNPTSPTHPL

The five most frequently occurring phage were then individually testedfor their affinity to TRAP using a modified ELISA method. In this assay,immobilized TRAP was probed with complete phage that were thenvisualized with a secondary antibody to the phage coat. The binding wascompared to wild type phage containing no expressed oligopeptides. Thesedata are presented in FIG. 4. The five phage with the highest consensusfrequency demonstrated at least a 40 fold increase in affinity for TRAPover wild type phage with Clone 5 phage showing a greater than 55 foldincrease in affinity (although not statistically significantly differentfrom the other phage). Clone 5 phage also possessed the highestconsensus frequency.

The actual affinity of Clone 5 phage for TRAP was determined in an aScatchard-type experiment utilizing immobilized TRAP and increasingconcentrations of phage particles. These data are presented in FIG. 6.They show that Clone 5 phage have an extremely high affinity for theTRAP substrate with a Kd of 1.1×10-10M.

Confirmation that Clone 5 phage were actually binding to TRAP wasobtained with Far-Western technology (FIG. 7). In these experiments, thetarget molecule, TRAP, as well as control proteins, bovine serum albumin(BSA) and RNase A, were separated with polyacrylamide gelelectrophoresis, transferred to a membrane and then probed with intactClone 5 phage. The only proteins in gels that were visualized appearedat the molecular weight for TRAP.

As TRAP is known to be present in resorption lacunae we sought todemonstrate that Clone 5 phage could recognize it on a resorbed bonesurface. Osteoclast lacunae (i.e. pits) were prepared on cortical bonewafers as described herein. These pits are a hallmark of authenticosteoclast activity and are frequently used to quantify bone resorptiveactivity. Osteoclasts were removed by gently scraping and washing thesurface of the bone wafers. The wafers were then probed with Clone 5phage. Visualization of the phage was with a secondary antibodyconjugated to HRP. Data showed that the localization of Clone 5 phagewas in resorption lacunae. Data showed that Clone 5 phage only bound tothe surfaces of resorption lacunae. These phage were visualized with aHRP-conjugated antibody directed against the phage (as in FIG. 7).Control experiments demonstrating no binding in unpitted wafers, withwild type phage and with the secondary antibody alone. Data showed thatTRAP can be removed from the lacuna by boiling and that phage will nolonger bind to these sites. Clone 5 phage localized only to priorresorbed osteoclast pits. There was no staining on adjacent surfaces.Moreover, there was no staining i) on un-pitted bone wafers, ii) withwild type phage, iii) when only the secondary antibody was used or iv)if the pitted bone wafers were boiled to extract all proteins notcovalently associated with the resorption surface. The staining in thepits was punctate in nature.

Data demonstrated that TRAP in osteoclast lacunae can be extracted andvisualized in a Far-Western with Clone 5 phage. In this experimentpitted and un-pitted wafers were extracted by boiling and the recoveredproteins separated on polyacrylamide gels. Typically far-Westernproteins were extracted from bone wafers. The only protein extractedfrom pitted bone wafers that could be visualized by Clone 5 phage wasTRAP. No material from unpitted wafers was visualized by Clone 5 phage.In gels, a 37 kDa protein was present in the 5 microgram lane, and morewas present in the 10 microgram lane, of pitted bone wafers. No band wasvisible in the 10 microgram lane of the unpitted bone wafers. A dosedependence for TRAP was also demonstrated as increasing itsconcentration led to more intense staining. No bands anywhere throughthe gel were visualized withS BSA or RNaseA. These data support theconclusion that Clone 5 phage were selected based on their affinityTRAP. After transfer to membranes protein bands were visualized asdescribed in FIG. 7. The only protein visualized was at the molecularweight for TRAP. This analysis was dose dependent since adding more ofthe protein extract increased the intensity of staining. Proteinsextracted from un-pitted wafers on which there was no osteoclastactivity were negative for staining with Clone 5 phage. Not only doesthis experiment demonstrate the specificity of Clone 5 phage for TRAP,it also demonstrates the feasibility of extracting detectable baitproteins from lacunae. This indicates that proteins can be recognized inthe lacunae.

The 12 amino acid oligopeptide corresponding to the sequence from Clone5 can inhibit osteoblast binding to osteoclast lacunae. FIG. 8 showsthat a synthetic peptide with the same sequence as in Clone 5 phage can,in a dose dependent manner, block osteoblast binding to pitted bonewafers. In these data we demonstrate that osteoblasts bind at anapproximately five fold higher level on bone wafers containingosteoclast pits as compared to un-pitted wafers. (For these experiments,approximately 20% of the area of the wafers was pitted.) A concentrationof 50 nM for the oligopeptide blocked osteoblast binding byapproximately 66%. A statistically significant inhibition for bindingoccurred down to a concentration of 10 pM of the oligopeptide. In datanot shown, we found that control proteins did not compete withosteoblast binding.

A FASTA protein data base search was performed using sequenceTPLSYLKGLVTV (SEQ ID NO:23) and identified four candidates with highhomology to this sequence. They are listed in Table 5. It is unlikelythat Rac1 (RAS-related C3 botulinum substrate) and Grin1 (glutamatereceptor) would have relevance to osteoblasts, however, Wnt7a (winglessdifferentiation receptor) and Gpc4 (glypican 4) may. Wnt7A is known toplay a key role in cell differentiation and has been associated withskeletal development but perhaps most importantly, Gpc4 is a member of afamily of attachment receptors expressed on the surface of cells. Itdoes not have a cytosolic domain and is believed to function solely incell attachment (reviewed in David, G., FASEB J. 7:1023-1030, 1993.).TABLE 5 FASTA Search for Clone 5 Homology Rac1 Ras-related C3 botulinumT P I T Y P Q G L A M A substrate : : . . : . : : . . . T P L S Y L K GL V T V Grin1 Glutamate receptor T P V S Y T A G F Y R I : : . : : : . .T P L S Y L K G L V T V Wnt7a Wingless-related MMTV K P L S Y R K P M DT D integration protein : : : : : . : T P L S Y L K G L V T V GPC4Glypican 4 I Y C S H C R G L V T V . . . : . . : : : : : T P L S Y L K GL V T V“:” indicates a perfect amino acid homology.“.” indicates an amino acid similarity

Glypican 4 (Gpc4) is one member of a close family of heparan sulfateproteoglycan-containing plasma membrane receptors found on fibroblasts,periodontal ligament cells and mesenchymal and marrow stem cells(Worapamorm, W., et al., Connective Tiss. Res. 41: 57-68, 2000;Siebertz, B., et al., Biochemical. J. 344: Pt3: 937-43, 1999.). Thesereceptors have been implicated in BMP signaling in Drosophila and ascytokine presenting receptors in bone marrow cells. Furthermore,mutations in the glypican family lead to a disease known asSimpson-Golabi-Behmel syndrome (Pilia, G., et al., Nature Genetics 12:241-247, 1996). Mice with this syndrome demonstrate a varied phenotype,however, the one common feature in all forms is skeletal abnormalities.

All of the analysis done for the clone 5, discussed herein, can beperformed on each and every molecule identified herein as having TRAPand/or osteoclast lacunae binding properties. Thus, the i) specificityfor TRAP with Far Western technology (as in FIG. 7); ii) determinationof a Kd for TRAP (as in FIG. 6); iii) examination of their localizationwithin resorption lacunae(as in FIG. 9); iv) examination of the effectof a synthetic peptide for interfering with osteoblast binding anddifferentiation (as in FIG. 8); v) identification of any differentiationeffects of these peptides; a FASTA search for their homology withproteins known to exist in nature; evaluation of positive homologouscandidates will be evaluated in a manner similar to glypican 4 (seeherein).

In order to demonstrate that GPC4 exists in osteoblasts and that itsfull length sequence also has affinity for TRAP, we performed amammalian two-hybrid study with a human TRAP cDNA and GPC4 obtained froma human osteoblast-like library (MG-63 cells). Individual transfectionof GAL-4-BD-TRAP, VP-16-AD-GPC4 or VP-16-AD-GPC412 into SaOS₂ cells didnot activate luciferase activity. However, co-transfection ofGAL-4-BD-TRAP and VP-16-AD-GPC4 into SaOS₂ cells demonstrated a 10-20fold increase in luciferase activity. Co-transfection of GAL-4-BD-TRAPwith a 12 amino acid deficient version of GPC4 (VP-16-AD-GPC412) alsoshowed no difference from control levels (FIG. 12). These datademonstrate that a full length fusion protein for GPC4 can interact witha fusion protein for TRAP and that the interaction depends on the same12 amino acids identified in the Clone 5 sequence. Moreover, these dataalso demonstrate that the osteoblast-like cell line, MG-63, containstranscripts for GPC4.

The 12 amino acid oligopeptide corresponding to the sequence from Clone5 can inhibit osteoblast binding to osteoclast lacunae. FIG. 13 showsthat a synthetic peptide with the same sequence as in Clone 5 phage can,in a dose dependent manner, block osteoblast binding to pitted bonewafers. In these data we demonstrate that osteoblasts bind at anapproximately five fold higher level on bone wafers containingosteoclast pits as compared to un-pitted wafers. For these experiments,approximately 20% of the area of the wafers was pitted. A concentrationof 50 nM for the oligopeptide blocked osteoblast binding byapproximately 66%. A statistically significant inhibition for bindingoccurred down to a concentration of 10 pM of the oligopeptide. In datanot shown, we found that control proteins did not compete withosteoblast binding.

c) Osteoblast cDNA Phage Display Library

A phage display library in M13 phage that expressed all the proteinsproduced by osteoblasts was made. The cDNA library was created from arat calvarial preparation containing 99.5% osteoblasts as judged byalkaline phosphatase histochemistry and osteocalcin immunocytochemistry(see Detailed Methods of Procedure). M13 phage were selected because oftheir larger capacity for expression constructs.

After three rounds of bio-panning, elution and amplification using TRAPas the bait we sequenced 25 clones. Of the clones with the highestconsensus sequence one of the sequences corresponded to TRIP, the TGFβreceptor interacting protein (Choy L. and Derynck, R., J. Biol. Chem.273: 31455-31462 (1998); Griswold-Prenner, I., et al., Molec. Cell.Biology 18: 6595-6604 (1998)). The true function of this protein isunknown, however, from reports in the literature it appears that ligandbinding to TRIP can either enhance or inhibit the effect of TGFβ.

In order to document that TRIP was, indeed, present in osteoblastsprimers from the known human/rat homologous sequences were synthesizedand osteoblast mRNA was PCR-probed. A prominent band representing theexpected 1 kB of sequence was obtained (Data showed that no band waspresent in the control lane, a small band was present in the 1 ug RNAlane and a heavy band was present in the 5 ug RNA laneS).

Functionality of TRIP for TGFβ signaling was determined by measuring theactivation of a TGFβ promoter/reporter construct, P3TP Lux (seeExperimental Design for a complete description of this construct). Thesedata indicate that TRAP binding to osteoblasts (presumably through TRIP)activates P3TP Lux (FIG. 9).

Thus, 1) An osteoblast cDNA phage display library identifies TRIP as aprotein with high affinity for TRAP, 2) TRIP is present in osteoblasts,and 3) TRAP activates TGFβ signaling pathways (presumably mediated byTRIP).

For our osteoblast binding and affinity assays single cell attachmenthas been measured. A single cell is captured by gentle suction on theend of a specially pulled glass pipette. It is then brought into contactwith a surface (that can be either organic or inorganic in nature) or amonolayer of cells (Typically a PC-3 cell was brought into contact witha bone wafer (i.e. the dark material on the left of each frame) for 120seconds. The cell was then pulled away from the wafer surface. The cellsdeformed during the pull-away, and the cell springing back toward thewafer after release of the suction in the pipette occurred. This serieswould represent “one” adhesive event. See FIG. 10 for quantitation ofthis effect.). Single cell-to-cell contact can also be performed bycapturing two individual cells and bringing them into contact with eachother. After defining two key variables that affect the adhesion, namelythe length of time of contact and the contact pressure (i.e. impingementforce), the occurrence of an adhesive event can be determined by pullingthe cell away from the surface. An adhesive event occurs when, underphase contrast microscopy, it is evident that the plasma membrane of thecell is deformed by its adhesion. For the purposes of this assay theaction of bringing a cell into contact with a surface for a specifictime and at a specific pressure as a “touch”. The adhesioncharacteristics of a cell for a surface was then determined by relatingthe number of “touches” to the number of adhesive events. (FIG. 10). Theeffect of different divalent cations on neutrophil attachment wasbmeasured. Both Mg+2 and Mn+2 created a conformational change in theintegrin receptor profile. Activation of these receptors leads toincreased affinity. Ca+2 had a minor effect on altering integrinconformation and the absence of divalent cations (EGTA) prevents anyneutrophil binding.

A second measure of cell affinity for a surface or a cell layer can bedetermined by measuring the force needed to remove the cell from thesubstrate. This is done by constructing a pipette that matches theapproximate diameter of the cell and manometrically measuring the forcenecessary to pull the cell away. Forces for this type of interaction arein the pico Newton range and can be determined

Methods such as these have been exploited by scientists for a number ofyears in the study of hematopoietic cell-cell interactions,cell-capillary wall interactions, cell-implant interactions, etc.(Artmann, G. M., et al., Biophysical Journal. 72(3):1434-41, 1997; Sung,K. L,. et al., Journal of Immunology. 158(2):919-27, 1997; Kwon, S. Y.,et al., Journal of Orthopaedic Research. 18(2):203-11, 2000; Olbrich,K., et al., Biophysical Journal. 79(1):321-7, 2000). The affinity ofosteoblasts for a bony surface and a resorption lacunae was disclosedherein as very “sticky” relative to control cells (i.e. lymphocytes,neutrophils, etc.).

It has been demonstrated that a number of lysosomal enzymes are used bythe osteoclast during bone resorption. Some of the more well studiedare: i) cathepsin B, ii) cathepsin G, iii) cathepsin L, iv) cathepsin Kv) lysozyme, vi) -galactosidase, and vii)-glucuronidase.

Examination of these enzymes as a “bait” for the phage libraries willallow us to determine if there is a common moiety on the enzymes forbinding (such as mannose-6-phosphate containing oligosaccharides) or ifany of the clones with high affinity for TRAP cross react with any ofthe other lysosomal enzymes. These experiments will be performed usingboth the random phage display library and the osteoblast cDNA phagedisplay library. The methods we will employ are those used to generatethe data in the Preliminary Studies section. Molecules other thanlysosomal enzymes may remain (or become exposed) on the resorptionsurface.

d) Identification of Phase Clones With High Affinity Binding toAuthentic Resorption Lacunae

The phage display technique can be used to identify molecules thatinteract with authentic resorption lacunae. The unique nature of thistechnique makes these experiments feasible even though both the libraryand the “bait” are unknown variables. That is, with either a randomphage display library or an osteoblast cDNA phage display library it isnot required to know which phage clones will show an affinity for aparticular substrate. Moreover, one does not need to know what thesubstrate will be. Nevertheless, phage with high affinity for resorptionlacunae are easily characterized and the corresponding binding substratecan be easily identified.

Specificity for the resorption lacunae was enhanced by removing themajority of library phage that demonstrate an affinity for bony surfacesthat are not covered with lacunae. This was achieved by firstsubtracting the phage in the disclosed libraries that have affinity forbone by exposing the libraries to un-pitted bone wafers. The phage titerfor the libraries used herein has been shown herein to be decreased byapproximately 25% when unpitted bone wafers are used as a subtractionsubstrate. Subtraction of phage library molecules that bind un-pittedbone wafers was obtained by three rounds of biopanning. Thus, minimalnon-specific binding will occur when pitted wafers are used as bait. Tooptimize selection, bone wafers with 40-60% of the area covered withosteoclast lacunae. Control experiments to be performed for thesestudies include: i) measuring the affinity of phage for resorptionlacunae after boiling or extraction of the bone wafer, ii) using wildtype phage to measure non-specific interactions, iii) demonstratinglocalization of phage within the lacunae as in FIG. 9

Upon identification of a phage clone with high affinity for a resorptionlacuna the nucleic acid from the enriched phage can be cloned andsequenced to identify the structure of the molecules recognizingresorption lacunae and pitted bone wafers. All of the characterizationdisclosed herein of identified molecules can be performed on themolecules identified in these selection activities.

The “bait” protein can be identified by a co-precipitation technique.Precipitation of a phage clone carrying its bait protein is accomplishedwith polyethelene glycol precipitation (PEG) (50). A protein extractfrom pitted bone wafers can be prepared by either boiling (as in FIGS. 9and 10) or gentle detergent treatment. The selected phage can then beadded to the extract, allowed to incubate and cleared from the solutionby stepwise addition of PEG. Recovery of the phage by centrifugation andelution of the bait protein can be followed by electrophoreticseparation (Schmaljohn C. et al., Virology. 258(1):189-200, 1999). Ifthere is more than one protein band that is eluted from the phage themajor substrate can be identified with Far Western technology as shownin FIGS. 8 and 10. The protein band can then be sequenced. This willgive information about which molecule in the resorption lacuna wasacting as bait for the particular phage.

e) Manipulation of TRIP and GPC-4 Expression—“Gain of Function/Loss ofFunction”

The levels of GPC-4 and TRIP gene products can be manipulated. This canshow that the gene products to demonstrate that they influenceosteoblast differentiation (using reporter systems) and binding toresorption surfaces (using a high resolution imaging andmicromanipulator system).

(1) TGFβ Signaling Pathways

TGFβ signaling evokes a number of complex and sometimes qualitativelydifferent responses in cells from the skeleton. TGFβ can be adifferentiating agent in growth plate chondrocytes and conversely it canstimulate proliferation and prevent differentiation in sternal andproliferating chondrocytes. In osteoblasts TGFβ has been shown to bothincrease and decrease alkaline phosphatase levels depending on thecofactors present, the cell line used, the substrate on which the cellsare grown and the number of passages for the cells (Kassem M., et al.,European Journal of Clinical Investigation. 30(5):429-37, 2000; YamadaT., et al., Histochemical Journal. 31(10):687-94, 1999; Chung C Y., etal., Biochemical & Biophysical Research Communications. 265(1):246-51,1999; Cheifetz S. et al., Connective Tissue Research. 35(1-4):71-8,1996; Harris S E., et al., Journal of Bone & Mineral Research.9(6):855-63, 1994; Bonewald L F., et al., Bone & Mineral. 17(2):139-44,1992). Disclosed herein, TGFβ stimulates alkaline phosphatase activityfrom freshly isolated osteoblasts. This observation supports thefollowing analysis.

Disclosed herein, TRAP can activate TGFβ signaling (FIG. 9). This wasdemonstrated with a TGFβ promoter/reporter construct that isspecifically sensitive to the Smad3/Smad4 transcription factor complex.It was also shown that TRAP can stimulate osteoblast differentiation asmeasured by a stimulation in alkaline phosphatase activity (FIG. 4).This is consistent with TRAP stimulation being mediated by TRIP and theSmad3/Smad4 transcription factor complex.

TGFβ activation pathways have been elucidated and are presented in FIG.11. Three main pathways arrive at two transactivating transcriptionfactor complexes: the JNK pathway, p38 pathway and Smad pathway. Thesethree routes of signaling have been highlighted in the figure.

Two potent inhibitors of the p38 pathway exist. They are RWJ65657 andSM203580 and can be used to identify this pathway.

Binding of TGFβ to its type II receptor recruits and phosphorylates thetype I receptor. The activated type I receptor then phosphorylates Smad3which can, in its activated state, bind to Smad 4. This complextranslocates into the nucleus where it interacts with other co-factorsto elicit gene transcription. The other two pathways rely on MEKK/JNKand p38 phosphorylation steps to activate ATF-2, another TGFβtranscription factor. It is shown herein that the Smad pathway (P3TPlux)can be activated by TRAP (FIG. 9) and it can be determined whether TRAPcan utilize either (or both) the JNK and p38 pathway. This can beaccomplished with a ATF-2 reporter construct (see Detailed Methods ofProcedure) and two specific inhibitors of the p38 pathway, namely,RWJ67657 and SB 203580. Both of these compounds have been gifted to us(by Johnson & Johnson and Smith Kline Beecham, respectively). They actas potent inhibitors of the p38 kinase (Barancik M., et al., Journal ofCardiovascular Pharmacology. 35(3):474-83, 2000; Tindberg N., et al.,Neurochemical Research. 25(4):527-31, 2000).

Osteoblasts can be transfected with the ATF-2 reporter and examine theeffect of both TGFβ signaling (positive control) and TRAP signaling.Dose response experiments with both TGFβ and TRAP will be performed todetermine the maximum effective concentration. Subsequently, this can berepeated in the presence of either RWJ67657 or SB203580, again over arange of doses. Toxicity of the compounds will be monitored with a MTTassay. The sub-toxic dose response curves, when compared to controlcurves, will reveal to what extent the JNK pathway is utilized by TGFβand TRAP in osteoblasts. That is, if complete inhibition of ATF-2signaling occurs with either of the inhibitory compounds then it can beassumed that little or no regulation occurs through the JNK pathway. Ifincomplete inhibition occurs then the fraction of signaling utilized theJNK pathway can be determined.

A dominant negative Smad3 construct (see herein) can be used todetermine to what extent the JNK/p38 pathway or the Smad pathway playsin the actual alteration of the osteoblast phenotype. The Smad pathwaycan be completely blocked using these techniques. A normal stimulationof alkaline phosphatase under these conditions would argue for theJNK/p38 pathway as being the dominant mechanism for changing thephenotype. The signaling experiments with the ATF-2 reporter (describedherein) indicate which pathway (i.e. JNK or p38) was dominant.

The first approach has been utilized in the literature to investigatethe interaction of TRIP with the type II TGFβ receptor (Choy L. andDerynck, R., J. Biol. Chem. 273: 31455-31462 (1998)). The strategy forthese experiments is to transfect TRIP-negative cell lines with theconstruct and examine the effect of TRAP on TGFβ signaling. ThreeTRIP-negative cell lines have been used in the past. They are HaCaT, akeratinocyte cell line, COS-1 cells and Mv1Lu (mink lung cells). (It isof interest that the mink lung cell line used for so many years to assayTGFβ levels is TRIP-negative). In principle co-transfection assays willbe performed (see Detailed Methods of Procedure) for both TRIP andeither the ATF-2 reporter or P3TPlux (the Smad reporter). The resultingcells will be exposed to TGFβ as a positive control to determineresponsiveness and gauge the level of reporter activity. They will thenbe exposed to TRAP as in FIG. 9. The effect of TRAP (both alone and incombination with TGFβ) will then be used to draw a conclusion aboutwhether TRAP activation of the cells is mediated through TRIP.

A mammalian two hybrid system can be used to show the interaction ofTRAP with TRIP (Sugawara T., et al., Endocrinology. 141(8):2895-903,2000; Shioda T., et al., Proceedings of the National Academy of Sciencesof the United States of America. 97(10):5220-4, 2000;Tagami T., et al.,Biochemical & Biophysical Research Communications. 253(2):358-63, 1998),as it was described herein for GPC-4. Much of the material is availablein kit form. In principle, GB133 cells containing a green fluorescenceprotein (GFP) gene under the control of a galactosidase promoter can bepurchased and used as the starting cell type. This cell is easily stablytransfected with a GAL4-fusion protein construct. GAL4 binds to thegalactosidase promoter and the fusion protein is the “bait” protein ofinterest. The cells are then transiently transfected with a protein ofinterest in a construct containing the EBNA-1 transactivating domain. Ifthe two target proteins interact, transcription of GFP proceeds and theproduct can be easily detected by fluorescence microscopy or UVspectroscopy. All of the materials needed to produce the constructs andeffect the transfection are available from CLONETECH and Stratagene.

f) Up Regulation of GPC4 and TRIP

Constructs containing GPC4 exist for transient transfection (Kleeff J.,et al., Pancreas. 19(3):281-8,1999; Song H H., et al., Journal ofBiological Chemistry. 272(12):7574-7, 1997; Steinfeld R., et al. Journalof Cell Biology. 133(2):405-16, 1996. which are herein incorporated byreference at least for the material related to GPC 4 constructs). Theseconstructs can be engineered to contain the enhanced green fluorescentprotein gene (GFP) (typically upstream). The eGFP plasmid is availablefrom CLONETECH in the form of a CMV driven internal ribosomal entry siteconstruct (P-CMV-IRES-eGFP). Cloning of the glypican gene into thisplasmid will allow for the expression of glypican-4 as well as eGFP fromthe same DNA. The internal ribosome entry site (IRES) allows the cellsto translate both the inserted gene (glypican-4) and eGFPsimultaneously. Thus, any cell glowing green under UV light should alsobe transfected with glypican-4. GFP-positive cells can be selected,one-by-one, for examination in the single cell attachment system. Datagenerated as in FIG. 10 will be used to demonstrate an effect ofglypican-4 on attachment.

In addition to osteoblasts, glypican-negative cells can also betransfected. For example, neutrophils and two prostate cancer cell lines(DU-145 and LnCap) have previously been shown not to have affinity forbone or resorption surfaces (Lewis, G. D., et al., Trans. Orth. Res.Soc. 23:161, 1998). Positive results with these experiments woulddocument the independence of glypican-4 in attachment to bone.

Controls include transfection with only eGFP, extraction of resorptionsurfaces (see FIGS. 9 and 10) and addition of heparan sulfate to thebinding medium. (Glypican-4 is a heparan sulfate proteoglycan andheparan sulfate will compete for binding sites.)

For “loss of function” experiments for glypicans there does exist aneffective antisense construct (Kleeff J. et al., Pancreas.19(3):281-8,1999; Kleeff J. et al., Journal of Clinical Investigation.102(9):1662-73, 1998) and anti-sera. This construct can be used upstreamof an IRES for eGFP (as described above for expression of glypican-4).Glypican protein levels will be assayed with ELISA's and correlated withthe extent of GFP production. Cells can be selected on a one-by-onebasis with fluorescence microscopy for use in the attachment studies.Direct experiments with anti-sera to glypican-4 can also be performed.

After expressing GPC-4 and TRIP on exogenous vectors in cells includingosteoblasts, upregulation of cellular binding to osteoclast lacunaeoccurs in GPC4 transfected cells and TGHβ signaling pathways areupregulated in TRIP transfected cells. These transfected cells would besuitable for ex vivo therapies which involve placing the transfectedcells into contact with bone. This could occur for example duringsurgery to repair a break in the bone.

A commercially available cloning system from Life Technologies (adivision of Invitrogen corp. www.lifetech.com) was used to clone boththe GPC4 and TRIP sequences into multiple different vectors. Thesevectors can be found in the Clonetech vector catalog, which isincorporated by reference herein for vector material. The system uses aphage recombination reaction rather than restriction endonucleases andligase. Recombination by phage occurs during the lytic phase of phagegrowth at specific DNA recombination sequences (ATT). Specific proteinsthat mediate the phage recombination are provided by Life Technologies.One series of reagents is used to move a particular sequence into an“expression clone”. A second series of reactions moves the sequence fromthe “expression clone” to an “entry clone”. The recombination reactionsare equivalent to highly specific cutting and ligation reactions butthey are improved because they are perfectly conserved and there is noneed for the synthesis or loss of nucleotides. Although, generalrecombinant biotechnologies involving for example cutting and ligationcould also be used, which are known to those of skill in the art.

Expression plasmids containing a strong promoter (CMV promoter) upstreamof either GPC4 or TRIP sequence which was upstream of an IRES sequenceand green florescent protein were constructed. The IRES sequence standsfor internal ribosomal entry site and the green flourescent proteinfluoreces under UV light. The reason for this construct was to produceboth the GPC4 or TRIP along with the green fluorescent protein on thesame gene in cells. Thus, cells glowing green under UV light indicatethat the GPC4 or TRIP had also been expressed. This is because thesesequences were upstream of the green fluorescent protein. Theseexperiments gave “transfection efficiency” of the system. The efficiencyapproached 50%.

Under the influence of the CMV promoter high levels of GPC4 and TRIP canbe produced. Tissue specific promoters to over-express and under-expressthe genes can also be used and are known in the art. This will allowcontrol of the molecules selectively in osteoblasts. Tissue specificpromoters used in the literature for these types of experiments are theosteocalcin promoter and the cbfa1 promoter. Both of these genes areonly expressed in osteoblasts.

In the next series of experiments the same cloning techniques were usedwithout the green fluorescent protein. In these experiments it was shownthat expression of GPC4 or TRIP would alter the behavior of our cells asexpected. That is, expression of GPC4 in GPC4-deficient cells wouldenhance their binding to bone and expression of TRIP in TRIP-deficientcells would activate the TGF beta signaling pathway. A number ofdifferent cell types have been tested, such as fibroblasts, prostatecancer cells, fibroblast cell lines, osteoblast cell lines and freshlyisolated osteoblasts

Transfection of bone cells ex vivo and then re-implantation of the cellsinto humans has been performed. Evans C H. Ghivizzani S C. Oligino T A.Robbins P D. Future of adenoviruses in the gene therapy of arthritis.Arthritis Research. 3(3):142-6, 2001 and Ghivizzani S C. Oligino T J.Glorioso J C. Robbins P D. Evans C H. Gene therapy approaches fortreating rheumatoid arthritis. Clinical Orthopaedics & Related Research.(379 Suppl):S288-99, 2000 have shown that ex vivo gene therapy forspecific molecules may be an effective way to treat musculoskeletaldiseases such as rheumatoid arthritis. The same strategy fortransfecting human osteoblasts with either GPC4 or TRIP can be used toaugment bone formation at fracture sites, around implants, at bonegrafting sites and systemically in osteoporosis patients. Technologyexists to achieve a high efficiency transfection and to re-implant cellsat specific sites.

g) Osteoblast Gene Expression Profile Assay

Both GPC4 and TRIP have been used in the creation of an osteoblast geneexpression profile assay. This assay utilizes cDNA primer pairs that canbe used to amplify a number of osteoblast gene products with real-timePCR. In addition to GPC4 and TRIP the following are assayed; osteocalcin(a bone specific protein), alkaline phosphatase (an enzyme present inlarge amounts in osteoblasts, cbfa1 (a transcription factor that inducesosteoblast differentiation), PTH receptor 1 (the receptor responsiblefor PTH action), PTH receptor 2 (a secondary receptor that may beresponsible for PTHrP action).

Results from these assays demonstrated that lead, which is known to havean adverse effect on bone formation, depresses all of the above geneproducts including GPC4 and TRIP.

The data are interpretted by analyzing an increase in PCR cycle number,i.e. the number of PCR cycles necessary to obtain product. The higherthe cylce number the smaller amount of starting mRNA. Table 6 displaysrepresentative data. TABLE 6 Control PCR cycle number Lead PCR cyclenumber GPC4 19 24 TRIP 21 26 osteocalcin 23 26 alkaline phosphatase 2327 cbfa1 24 28 PTH receptor 1 28 33 PTH receptor 2 31 32

These data demonstrate that the heavy metal ion, lead, can alterosteoblast gene expression. It also suggests that other regulatoryagents will also be able to modify these genes. Most importantly, thedata validate the importance of GPC4 and TRIP as participants indefining the osteoblast phenotype.

2. Example 2 Detailed Methods of Procedure for Example 1 a) Formation ofOsteoclastic Resorption Pits

Bovine diaphyseal cortical bone wafers (4×4×0.3 mm) cut with a low speeddiamond saw (Buehler, Evanston, Ill.) are used as a substrate forosteoclastic resorption. Wafers are placed into 70% ethanol and cleanedby ultrasonication for 15 min followed by multiple rinses in sterile PBSand sterile water. Bone wafers are dried and stored at −20° C.

Osteoclast containing cell preparations are obtained from 4-6 day-oldeuthanized rat pups. Long bones (femurs and tibias) are removed, freedof adherent soft tissues, and curretted with a scalpel blade in 1.0ml/animal isolation media, pH 7.2.(Minimal Essential Medium+Earles salts(Gibco, #51200), buffered with 20 mM HEPES containing Nonessential AminoAcids, L-Glutamine, heat-inactivated Fetal Bovine Serum-10%, andPenicillin/Streptomycin). The cell suspension is triturated with apipette (10-20×) followed by a 10 s settling period for larger pieces tosediment. Supernatant cell suspension is then removed and aliquoted into100 ul portions to be added to a 96 well culture dishes containing thewafers (one wafer/well). Bone wafers are pre-wetted with the aboveisolation medium. The cell suspension is allowed to incubate with thebone wafers for exactly 20 min at 37° C. Wafers are then washed in warm,sterile PBS for 4-6 seconds, and placed in incubation medium (isolationmedium minus HEPES) containing 1×10-8 M PTH, pH 7.25, (150 ul/slice).Scanning electron micrographs of one of these osteoclasts resorbing acortical bone wafer were performed. Since there is no immune system inthese cultures, we have found no problem with rat osteoclasts resorbingbovine bone. Typically the cell created two osteoclast lacunae (pits) ina cortical bone wafer.

b) Quantification of Number and Extent of Resorption Lacunae:

The entire bone wafer was digitally photographed at 200× magnificationusing a JVC TK-1070U video camera and Olympus BH-2 microscope attachedto a Macintosh IIci computer with a Colorsnap frame-grabber board. Thenumber and area of osteoclast lacunae are quantified with Osteometrics®software (Osteoclast lacunac were stained. The outlines of the lacunaeare traced (dotted lines). The number of pits, total area resorbed andthe area per pit are calculated.). A minimum of six wafers is used toanalyze each concentration of factor or cell culture.

c) Osteoblast and Osteoprogenitor Cell Isolation

Thin plates of bone from the parietal segments of the neo-natal ratcalvaria are dissected by first removing the entire calvarium from therat pups and then trimming away the unwanted tissue. The procedure isperformed aseptically.

The enzymatic digestion of these fragments is performed by the followingmethod: During dissection the parietal fragments (2/calvarium) arestored in a shallow pool of isolation buffer (see below) in a plasticculture dish. The fragments are then incubated in a buffer (2.5calvaria/ml) containing bacterial collagenase (Sigma, 0.5 mg/ml) for atotal of 100 minutes. The incubation vessel is a polypropylene 50 mlbeaker in a 37° C. oscillating water bath (1 Hz). Cells are collected ateach of five 20 minute intervals (Fractions 1-5) by decanting the enzymecontaining solution into tubes and centrifuging for 3.0 minutes at500×g. Digestion for longer periods of time does not release any morecells. During the centrifugations the calvarial fragments are returnedto the 37° C. water bath (without shaking) to keep them at constanttemperature. The supernatant enzyme solution is then returned to thecalvarial fragments for the next 20 minute digestion. The cell pellet iswashed by resuspension and centrifugation in the isolation buffer. Cellcounts in each period are performed with a hemocytometer. Cellsrecovered from the early periods are stored in isolation buffer at 37°C. until the digestion of the calvarial fragments is complete.

Isolation buffer is composed of: 25 mM HEPES, 10 mM NaHCO3, 100 mM NaCl,3 mM K2HPO4, 12 mM mannitol, 24 mM KCl, 1 mM CaCl2, 5 mg/ml glucose, 2mg/ml bovine serum albumin, 100 units/ml penicillin, 100 ug/mlstreptomycin, pH 7.4.

The collagenase enzyme is obtained by screening a number of preparationsfrom Sigma. The criteria for selection is 1) minimal cell damage(assessed microscopically), 2) maximum yield of cells per calvarium and3) greatest viability after plating. The chosen lot of enzyme is thenpurchased in quantity and further treated with N-tosyl-L-lysinechloromethyl ketone (TLCK), a clostripain inhibitor. The treatmentconsisted of a 20 minute incubation of the enzyme with the TLCK withsubsequent extensive dialysis against water at 4° C. The enzymepreparation is then lyophilized and stored at −20° C. in 500 ugaliquots.

Culture of the cells is by standard cell culture techniques. Usually thecells from each of the five fractions are diluted to 25,000 cells/ml inculture medium and plated at 125 cells/mm2 in 16 mm tissue culturemultiwells.

The culture medium consists of a balanced salt solution containing MEMamino acids (Gibco), and BGJ vitamins (Gibco). Penicillin (100 units/ml)and streptomycin (100 ug/ml) are also added to the medium. For mostexperiments the culture medium is supplemented with dialyzed fetalbovine serum protein at 2.0 mg/ml. The serum protein is prepared bydialyzing (10-12,000 MW cut-off) commercially available fetal bovineserum (Gibco) against many changes of water at 4° C. followed bylyophilization.

d) Cell Adhesion to Bone Matrix Surfaces

Transverse slices of bovine femoral diaphyseal cortical bone (4×4×0.3mm), cut with a low speed diamond saw (Buehler, Evanston, Ill.) andsterilized by ultrasonication, served as the substrate for cellattachment studies. Confluent cells were radiolabeled with 3H-thymidine(5 Ci/ml) for 24 hours followed by a cold thymidine chase (0.1 mM) for30 min. The cells were removed from the culture dish with trypsin/EDTA(5 min. incubation), centrifuged at 1500 rpm for 5 min. and the pelletresuspended in 4.0 ml of their respectivemedia's+Penicillin/Streptomycin. The cells were then counted, diluted to20,000-100,000 cells/ml, and the specific activity was determined (i.e.CPM/cell). 4000-20,000 cells (2001) were subsequently added to eachwafer and allowed to incubate (37° C., 5% CO2) for exactly 10 min. Thewafers and cells were then washed with gentle agitation. The extent of3H-thymidine label remaining on the wafers was measured in ascintillation counter. The number of cells attached to the wafer surfacewas calculated from the specific activity.

e) DNA Constructs

Wild-type and dominant negative mutant cDNA (C-terminal truncation, c)of human Smad 1-3 were a gift from Dr. Rik Derynck, and subcloned intothe mammalian expression vector pCMX and into the replication competentavian sarcoma retrovimis RCASBP(A). Wild-type Smad1-3 and dominantnegative Smad1-3c sequences were verified following subcloning usingautomated sequencing. The TGF-responsive p3TP-Lux reporter construct andthe dominant negative type II TGF receptor was a gift from Dr. JoanMassagué.

f) Transient Transfection and Luciferase Assay

Osteoblasts, cultured at 30-40% confluence in 6-well plates, aretransfected on day 2 after plating using the transfection reagentSuperfect (Qiagen Santa Clarita, Calif.) according to the manufacturer'sguidelines. Individual experiments are internally controlled for amountof total plasmid DNA, with equal amounts of p3TP-Lux reporter, controlSV40-renilla plasmid for normalization of transfection efficiency, andtarget construct and/or vector control DNA for all co-transfectionexperiments. Following transfection, the cells are placed in mediacontaining DMEM, Penicillin/Streptomycin, and 10% NuSerum IV. After 12hours, they are incubated for 6 hours in serum-free media (containingDMEM, hyaluronidase 4 U/ml, Penicillin/Streptomycin, and supplementedwith 10 pM triiodothyronine (Sigma, St. Louis, Mo.), 60 ng/ml insulin,and 1 mM cysteine (Sigma, St. Louis, Mo.)) plus the factor or cytokineof interest. Eighteen hours later, the cells are harvested and assayedfor luciferase activity using the Promega dual luciferase assay system.Renilla luciferase values were used to normalize each sample fortransfection efficiency.

g) Western Blot Analysis

Proteins will be separated by SDS-PAGE and transferred tonitrocellulose. The blot will be stained with Ponceau S and thepositions of the molecular weight standards marked. The blot will beblocked sequentially with solution 1 (1×PBS, 3% BSA, 0.05% Tween-20) andsolution 1/10% normal goat serum (NGS). Primary antibody in solution1/10% NGS will be incubated for 3 hrs with rocking at room temperature.The blot will be washed and secondary HRP-goat anti-rabbit in solution1/10% NGS added for 45 min. The blot will be developed with 6%4-chloronapthol in 20% methanol, 1×PBS, 0.036% hydrogen peroxide.

h) Screening of Phage Binding to TRAP by Phase ELISA, Far-Western andScatchard Analyses

Small-scale phage preparations, obtained from single colonies of thethird round of affinity bio-panning were analyzed for binding to TRAP byphage ELISA. Briefly, in this method, selected phage at increasingconcentrations were incubated for 2 hr at room temperature in TRAP orBSA-coated wells. Phage that bound to immobilized TRAP were detected byincubation with HRP-conjugated anti-M13 antibody (Pharmacia#27-9411-01), followed by incubation with HRP substrate (ABTS Sigma#A1888) and read at an OD of 410 nm.

A Far-Western technique was used to document that the selected phagewere indeed binding to TRAP. In this procedure, increasingconcentrations of TRAP (1-4 ug) and control proteins (BSA and RNase, 5ug) were loaded in a 10% SDS-PAGE gel and electrophoresed. They werethen transferred to PVDF membranes (NEN) and incubated with 10¹⁰ phageparticles from selected clones at 4° C. for overnight. The membrane waswashed in PBS with 0.5% [v/v] Tween 20 four times. An anti-M13 phageperoxidase-conjugated antibody at a dilution of 1:15000 was added andgently swirled at room temperature for an hour. In the last washingprocedure, the membrane was incubated in PBS without Tween 20. Detectionof the phage/antibody complex was accomplished using ECL-plus (Amersham)with the membrane being exposed to Kodak Biomax MR film for 30 seconds.

Phage affinity for TRAP was determined with a modified Scatchardanalysis. In this analysis 96 well plates coated with a fixed amount ofTRAP (10 ng/ml) were incubated with a serial dilution of phage (titerfrom 10¹²/ml˜10⁹/ml). After a one hour room temperature incubation, eachwell was washed with PBS with 0.5% [v/v] Tween 20 four times. The boundphage where then amplified directly in the wells by the addition of 100ul of log-phase ER2537 (M13 bacteria host). The number of phage in eachwell was then determined with a standard plaque assay. For thenon-specific binding estimation, the experiment was repeated in thepresence of a 100 fold excess of TRAP. The bound and free fraction ofphage were then determined.

i) Mammalian Two-Hybrid Assay

A mammalian two-hybrid assay was performed utilizing materials obtainedfrom Clonetech (catalog #K1602-1). In this procedure we created twofusion proteins; a GAL4-BD-TRAP and a VP16-AD-GPC4. That is, TRAP wasinserted into a plasmid containing a GAL-4 BD (binding domain) andglypican 4 (GPC4) was inserted into a plasmid containing the VP-16-AD(activation domain). The TRAP sequence was obtained from Dr. JamesBixley, University of Missouri, Columbia, Mo. GPC4 was obtained from ahuman osteoblast cDNA library from MG-63 cells.

GAL-4-BD-TRAP binds to the promoter of a luciferase reporter gene andthe VP-16-AD-GPC4 activates RNA polymerase II activity. If the twotarget proteins interact with each other they will bring the bindingdomain protein into the vicinity of the activation domain and luciferasetranscription will occur. The presence of luciferase activity inco-transfected cells is evidence for a physical association between theproteins.

In a control experiment, a second VP-16-AD protein was constructed usinga GPC4 sequence deficient in the 12 amino acids identified from Clone 5phage biopanning. This construct is termed VP-16-AD-GPC412. Deletion ofthe 12 amino acids from the GPC4 gene was accomplished with linkerscanning mutagenesis

Pairs of plasmids were co-transfected into SaOS₂ osteoblast-like cellsusing Lipofectamine®.

j) In Situ Staining

To confirm that the phage recognized TRAP in a bone resorption lacunaewe performed a binding assay in osteoclast pits prepared on corticalbone wafers in vitro. In this assay, osteoclast lacunae were formed byculturing neo-natal rat bone marrow cell isolates on cortical bonewafers in the presence of PTH (1×10⁻⁸ M) for 10-14 days. The osteoclastswere removed by gentle scraping and washing. 10¹⁰ phage were then addedto each wafer and incubated for 2 hours at room temperature. The waferswere washed four times in PBS and visualized with a primary anti-phageHRP conjugated antibody as described in the Far-Western technologypresented above. For control experiments we removed TRAP from thelacunae by boiling the wafers for 2 minutes.

k) Competition Cell Binding Assay

To measure the effect of selected phage peptides on osteoblast bindingin osteoclast lacunae we performed a cell binding assay in the presenceof increasing concentrations of the phage peptide. Osteoblasts wereprelabeled with L-[4,5-³H]Leucine (250 μCi TRK636-250 μCi, Amersham )for 24 hrs. The osteoblasts were then added to pitted bone wafers in thepresence of increasing concentrations of a synthetic 12 amino acidpeptide that was identical to the phage sequence. After a two hourincubation at 37° C. the wafers were washed and the amount of radiolabelassociated on each wafer determined by scintillation spectrometry. Thespecific activity of the cells (i.e. cpm/cell) was used to determine thenumber of cells adhering to the wafers.

l) Statistics

All data are presented as the mean±one standard error of the mean.Statistical significance was determined by ANOVA.

m) MTT Cell Viability Assay

MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide)conversion to formazan is used as a test for cell viability. To make themeasurement, MTT is added to the cultures for 30 min and then the mediumis aspirated from the wells. The insoluble formazan reaction productremains intracellular in the viable cells. To solubilize the formazan,0.2 ml of isopropanol containing 0.04 N HCl was added to each well, andthe plate was spectrophotometrically analyzed in an ELISA plate readerat an absorbence wavelength of 570 nm. Cell number is determined fromsets of standard wells containing known numbers of cells.

n) DNA Assay and Thymidine Incorporation

Total DNA is measured with an assay specifically designed forquantification of small numbers of cells in culture. In this assay theculture medium is removed and the cells rinsed with 1 ml of 0.15 M NaCl.The cells are removed from the dishes by the addition of trypsin (0.05%)and EDTA (0.4 mg/ml) in a balanced salt solution containing 15 mM HEPESand no calcium or magnesium. The dishes are rinsed with an additional0.25 ml of the HEPES buffer and pooled with the first extract. Onemilliliter of culture medium containing 2 mg/ml BSA is then added andthe DNA precipitated by the addition of 0.5 ml of 50% trichloroaceticacid (TCA). The samples are chilled to 4° C. and centrifuged at 14,000×gfor 30 minutes. The supernatant is re-moved and the pellet rinsed with0.2 ml of 0.01 N potassium acetate in absolute ethanol at 4° C. Thesamples are then recentrifuged at 14,000×g for 10 minutes and theethanol aspirated. The samples are then dried at 50° C. Standards areprepared from calf thymus DNA and also dried. A solution of 2.0 Mdiaminobenzoic acid is pre-pared in double distilled water to which isadded 100 mg/ml activated charcoal. After stirring for 30 minutes, thesuspension is filtered through a 0.65 mM filter to remove the charcoal.0.1 ml of the resultant solution is added to each sample. Afterincubation for an additional 30 minutes at 50° C., 3 ml of 0.6 Mperchloric acid is added to the samples. The DNA is quantitated byfluorescence spec-trophotometry of the samples with an excitation at 408nm and emission at 500 nm. The sample values are calculated from alinear regression analysis of the DNA standards.

De novo DNA synthesis is measured with radiolabelled thymidineincorporation. In this assay the cells are exposed to 2.0 Ci ofradiolabelled thymidine (methyl-3H-thymidine, Amersham) at a finalconcentration of 5.0 M for two hours. Following the exposure, theradioactive medium is discarded, the cells removed, and the DNA acidprecipitated by the addition of perchloric acid. Followingcentrifugation the supernatant is discarded and the pellet isredissolved in NaOH. Acid-insoluble radioactivity is determined byliquid scintillation spectrometry. Standards of the radiolabelled mediumare prepared for the direct estimation of the incorporation offemtomoles of thymidine into DNA.

Detailed methods have been previously published for: ELISA (Blaine T A,et al., J Bone Joint Surg 78(A):1181-92, 1996; Blaine T A, et al., JBone Joint Surg 79(A):1519-28, 1997; Pollice P, et al., J Orthop Res16:607-704, 1999), Northern blot (Blaine T A, et al., J Bone Joint Surg78(A):1181-92, 1996; Schwarz E M, et al., Proc Natl Acad Sci USA90:7734-8, 1993), RNAse protection assay (Grimsrud C, et al., J OrthopRes 16:247-55, 1998), Western blot (Schwarz E M, et al., Genes Dev11:187-97, 1997), EMSA (Schwarz E M, et al., Proc Natl Acad Sci USA90:7734-8,. 1993; 78, 79), Luciferase reporter construct assays (SchwarzE M, et al., Proc Natl Acad Sci USA 90:7734-8, 1993),Immunohistochemistry and semiquantitative analysis (Schwarz E M, et al.,J. Orthop. Res. In Press 1999; Hicks D G, et al., J Bone Joint Surg78:482-96, 1996), Spleen cell osteoclast bone resorption cultures(Franzoso G, et al., Genes Dev 11:3482-96, 1997), Osteoclastogenesisassays (Franzoso G, et al., Genes Dev 11:3482-96, 1997; Takahashi N, etal., Endocrinology 122:1373-82, 1988), and Generation of stable celllines (Schwarz E M, et al., J Virol 72:5654-60, 1998) which are hereinincorporated by reference at least for the material related to themethods for which each was cited.

o) Vertebrate Animals

Sprague-Dawley derived white laboratory rats can be used. 5 timedpregnant female rats per week are preferred. The animals will be allowedto deliver their pups and can the 2-3 day old pups can be transportedthe to the laboratory for sacrifice and tissue preparation. Thecalvarial tissue of the pups will be the source from which we willisolated and culture cells from the osteoblast lineage. The long bone ofthe pups will be used for the isolation of osteoclasts.

Sacrifice of the 2-3 day old rat pups can be by any rapid decapitationafter sedation by exposure to a 100% atmosphere of carbon dioxide. Thelevel of sedation can be assessed by a pedal touch reflex prior tosacrifice.

3. Example 3 A Phage Display Technique Identifies a Regulator of CellDifferentiation a) Abstract

40 phage clones with very high affinity for TRAP were sequenced and ofthe clones with multiple consensus sequences we identified a regulatoryprotein that modulates osteoblast differentiation. This protein is the“TGFbeta receptor interacting protein” (TRIP-1). The data demonstratethat TRAP activation of TRIP-1 evokes a TGFβ-like differentiationprocess. Specifically, TRIP-1 activation increases the activity andexpression of osteoblast alkaline phosphatase, osteoprotegerin, collagenand Runx2. Moreover, it was shown that TRAP interacts with TRIPintracellularly, that activation of the TGFβ type II receptor by TRIP-1occurs in the presence of TRAP and that the differentiation process ismediated through the Smad 2,3 pathway. It is also demonstrated thatosteoblasts, when cultured in osteoclast lacunae containing TRAP,rapidly and specifically differentiate into a mature bone formingphenotype.

b) Introduction

The formation of new bone during the process of bone remodeling occursalmost exclusively at sites of prior bone resorption. As there are oneto two million active remodeling sites in an adult skeleton at any pointin time (Parfitt A. M., The physiologic and clinical significance ofbone histomorphometric data, in Bone Histomorphometry: Techniques andInterpretation. Robert R. Recker, ed., CRC Press, Boca raton, Fla.,1983, pp. 143-223), this spatial localization of formation plays a keyrole in maintaining skeletal architecture. Aberrant or disorganizedformation could lead to architectural changes that would weaken skeletalstructure.

Disclosed herein an osteoblast protein (TGFβ receptor interactingprotein, TRIP-1) possesses very high affinity for TRAP and is poised foractivating the TGFβ differentiation pathway in osteoblasts. TRIP-1 hasbeen previously described in other cell types and has been shown tomodulate TGFβ signaling in both a stimulatory and inhibitory fashion(Choy, L. and Derynck, R., J. Biol. Chem. 273: 31455-31462 (1998); Chen,R H., et al., Nature 377: 548-552 (1995)). In osteoblast systems TGFβsignaling pathways control osteoblast differentiation (Kassem M., etal., European Journal of Clinical Investigation. 30(5):429-37, (2000);Yamada T. et al., Histochemical Journal. 31(10):687-94, (1999); Chung CY. Et al., Biochemical & Biophysical Research Communications. 265(1):246-51, (1999); Cheifetz S., et al., Connective Tissue Research.35(1-4):71-8, (1996); Harris S E., et al., Journal of Bone & MineralResearch. 9(6):855-63, (1994); Bonewald L F. Et al., Bone & Mineral.17(2): 139-44, 1992). This effect is modulated by the interaction ofTRAP with TRIP-1.

c) Methods

Purified type V TRAP was obtained as a generous gift from Dr. R. M.Roberts, University of Missouri, Columbia, Mo. This enzyme, also knownas uteroferrin, shares identity with osteoclast TRAP (Ling P, Roberts RM, Journal of Biological Chemistry. 268:6896-902 (1993)).

Cortical bone wafers were obtained by cutting 4.0×4.0×0.3 mm sectionsfrom bovine femoral cortical bone obtained from a local abattoir.

Isolated osteoblasts were prepared from neo-natal rat calvaria aspreviously described (Martinez D A, et al., J. Cellular Biochemistry 59:246-257 (1995)). Alkaline phosphatase assays and standard Westernanalyses were performed as previously described (Ionescu A M. ET AL.,Journal of Biological Chemistry. 276:11639-47 (2001)).

(1) Construction of a T7 Primary Rat Osteoblast cDNA Library

A T7 phage display library of rat osteoblast cDNAs was constructed froman existing primary rat osteoblast day 8 cDNA plasmid library generatedfrom primary isolated rat osteoblasts. The cDNA inserts of the plasmidlibrary were excised by digestion with EcoRI and NotI and insertedbetween the corresponding sites of an equimolar mixture of T7Select 1-1vector arms (T7Select System Manual, Novagen). The resulting phagelibrary contained 5.6×10⁷ independent clones/mL, as determined by plaqueassays. The library was amplified once by infecting a mid-log-phaseEscherichia coli (BLT 5615 bacterial strain) culture (250 ml, OD₆₀₀ 0.6)with the phage library at a multiplicity of infection of 0.001. Aftercell lysis, the phage lysate was made in 0.5M NaCl, clarified bycentrifugation, and stored at minus 80° C. The insert sizes of 24individual clones as well as of the complete library were analyzed byPCR with the forward primer 5′-GGAGCTGTCGTATTCCAGTC-3′ and the reverseprimer 5′-AACCCCTCAAGACCCGTTTA-3′.

(2) T7 Phase Clone Biopanning Procedure

An aliquot of the amplified phages (10⁹ pfu) were allowed to bind toTRAP which was immobilized on an ELISA plate for 2 h while rotatinggently. Unbound phages were removed by washing ten times with 0.2 ml 1 MNaCl, 0.1% Tween-20 in PBS, pH 7.2, and further washed twice in 0.2 mlPBS and finally resuspended in 100 μl elution buffer (Novagen).

Ten microliters of the supernatant was used to determine the amount ofdetached phage in each round of selection. The remaining 90 ul of thesupernatant was added to a 10 ml culture (OD 0.6) of E. Coli (BLT5615).The bacteria had been induced with 100 μl of 100 mM IPTG 30 min beforephage addition, to ensure production of the phage capsid protein.Approximately two hours after phage addition the bacteria were lysed andthe phage sublibrary was added to the ELISA plate (Nune, Rochester,N.Y.) coated with TRAP. After binding and washing the sublibrary, a newround of selection was started. Following two rounds of selection, 40plaques were arbitrarily isolated from LB plates and each dissolved inphage extraction buffer (100 mM NaCl, 20 mM Tris-HCl pH 8.0 and 6 mMMgSO₄). In order to disrupt the phages, the dissolved material was mixed1:1 with 10 mM EDTA pH 8.0 and heated at 65° C. for 10 minutes. Thephage DNA was then amplified by PCR, using T7 SelectUP and T7 SelectDOWNprimers (T7Select Cloning kit, Novagen). After amplification, the PCRfragments were purified by adding 1 ml 100% ETOH to precipitate the PCRproduct. The purified PCR fragments were then sequenced using ABI 377Big Dye autosequence kit (Applied bioscience). Based on the sequenceresults, the predicted amino acid sequence displayed on the T7 phagecapsid can be determined. The candidate clones were amplified and usedto check the affinity to TRAP with an ELISA method.

(3) Phage ELISA and Far-Western

Small-scale phage preparations, obtained from single colonies of thethird round of affinity bio-panning were analyzed for binding to TRAP byphage ELISA. Briefly, in this method, selected phage at increasingtiters were incubated for 2 hr at room temperature in TRAP or BSA-coatedwells. Phage that bound to immobilized TRAP were detected by incubationwith HRP-conjugated anti-T7 antibody (Novagen), followed by incubationwith HRP substrate (ABTS Sigma A1888). The absorbance was read at an ODof 410 nm.

A Far-Western technique was used to document that the selected phagewere indeed binding to TRAP. In this procedure, two concentrations ofTRAP and control proteins (BSA, 5 ug) were loaded in a 10% SDS-PAGE geland electrophoresed. They were then transferred to PVDF membranes (NEN)and incubated with 10¹⁰ M13 phage particles from a GPC4 phage (Clone 5)which have been shown to have high affinity and high specificity forTRAP. The membrane was washed in PBS with 0.5% [v/v] Tween 20 fourtimes. An anti-M13 phage peroxidase-conjugated antibody (AmershamPharmacia) at a dilution of 1:15000 was added and gently swirled at roomtemperature for an hour. In the last washing procedure, the membrane wasincubated in PBS without Tween 20. Detection of the phage/antibodycomplex was accomplished using ECL-plus (Amersham) with the membranebeing exposed to Kodak Biomax MR film for 30 seconds.

(4) OPG Sandwich ELISA

ELISA plates were coated by overnight incubation with 0.1 ml ofcarbonate buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, 0.02% NaN₃, pH 9.6)containing 1 μg/mL of anti-OPG antibody at 4° C. per well. The plateswere blocked with 0.2 ml of 5% dry milk in PBS per well for 1 hr at 37°C. 200 μl of medium prepared from different treatment cells as describedabove were added and incubated for 1 h at 37° C. Samples and serialdilution of OPG standards were loaded in triplicate to the plates (0.2ml/well). After washing with PBS-Tween (0.1%), the bound OPG wasquantified by successive incubation with another detection antibodyconjugated with biotin (1 hr each at 37 C). After incubation, the platewas washed with PBS-Tween for ten times and incubated with 100 ulStreptavidin with HRP-conjugated (1/10000 dilution from stock) for 30min at room temperature. After incubation, the plates were washed withPBS-Tween. 0.2 ml of 2,2′-azinobis (3-ethylbenzthiazolinesulfonic acid)solution (ABTS Sigma #A1888) per well was added for reaction withhorseradish peroxidase. The plate was then measured at 405 nm in anELISA reader.

(5) Glutathione S-Transferase (GST) Fusion Protein Preparation andPull-Down Assay

GST-TRAP and GST-TRIP fusion proteins, and GST control protein werepurified as instructed by the manufacturer (Amersham Pharmacia).Briefly, plasmids containing GST-fusion protein expressing cDNA weretransformed into a BL21DE3)pLysS bacteria strain and selected forampicillin and chloramphenicol resistant colonies. Selected colonieswere grown in LB medium at 30° C. until OD₆₀₀ reached 0.6 to 1. Then 0.1mM IPTG was added to medium for 3 hours. Bacteria were lysed by B-PER(Pierce) with 1 mM PMSF. Lysed bacteria were spun down and thesupernatants were collected. The GST fusion proteins were pulled down byglutathione coated beads (Amersham) in 4° C. for 1 h then washed threetimes with NETN buffer (20 mM Tris/pH 8.0, 100 mM NaCl, 6 mM MgCl₂, 1 mMEDTA, 0.5% NP-40, 1 mM DTT, 8% glycerol, and 1 mM PMSF). The purifiedGST fusion proteins and beads were suspended in 100 μl NETN buffer.Resuspended GST-proteins and beads were incubated with 5 μg purifiedTRAP or RIPA lysed transfected cell lysate. After incubating for 1 h at4° C. with agitation, the glutathione-coated beads were washed with NETNbuffer four times then the protein complexes were loaded in SDS-PAGE andvisualized by using the ECL-plus method.

(6) Mammalian Two-Hybrid Assay

Transfections were performed using the Panver LT-1 reagent method(Panvera) as described in the product sheet. Briefly, 1.5-3×10⁵ cellswere plated on 35-mm dishes for 24 h, and the medium was changed to DMEMcontaining 10% FBS 2 h before transfection. Cells were transfected with0.5 μg of plasmids expressing a Gal4-DBD (DNA binding domain) fused witha full-length TRIP-1 cDNA or an anti-sense TRIP-1 cDNA and a VP-16AD(activation domain) fused with a TRAP cDNA as indicated. A Gal4 responseelement controlled firefly luciferase expression plasmid, pG5-Luc, wasused as reporter gene. A Renilla luciferase expression plasmid pRL-SV40was used as an internal control for transfection efficiency. The totalamount of DNA was adjusted to 5 μg with pCMX vectors.

(7) Statistics

All data are presented as the mean±one standard error of the mean.Statistical significance was determined by ANOVA.

d) Results

Osteoblast differentiation at sites of bone remodeling is mediated by anumber of regulatory factors. One of the key factors is TGFβ. In mostsystems, including the typically the disclosed system, TGFβ is known tobe a potent enhancer of the osteoblast phenotype. FIG. 14 shows that inthe presence of TGFβ isolated osteoblasts show an increase in alkalinephosphatase activity, and an increase in Runx 2, osteoprotegerin (OPG)and collagen protein synthesis. In an exactly analogous fashion, TRAPdemonstrates the same effect. Control phosphatases such as myokinase andATPase have no effect on the cells.

The effect of TRAP on osteoblasts may be one mechanism by which thecells are induced to differentiate only at sites of prior boneresorption. In order to define what osteoblast proteins may be involvedin this mechanism immobilized TRAP was probed with T7 phage that wereexpressing proteins from an osteoblast cDNA library. After three roundsof bio-panning 40 phage clones with very high affinity for TRAP weresequenced and analyzed. Three of the clones contained sequences that maybe involved in osteoblast differentiation. They were type I collagen,Sox9, and TRIP-1. However, the sequences for type I collagen and Sox9were from the 3′ untranslated region of the messages. The TRIP-1sequence, however, was from the coding region of the protein.

FIG. 15 a demonstrates that the TRIP-1-expressing phage show adose-dependent affinity for TRAP. FIG. 15 b is a compilation of datathat indicate the binding of TRIP-1 to TRAP is specific and of highaffinity. For these experiments we prepared a plasmid construct withhuman TRIP-1 cDNA fused to glutathione-S transferase (GST-TRIP). As acontrol protein we utilized the GST vector alone. GST-TRIP and TRAP wereincubated for 1 hour and any proteins associated with the GST-TRIP wereextracted from the reaction mixture by incubation with glutathionecoated beads. The proteins were analyzed by Far-Western analysisutilizing a phage clone with high affinity for TRAP. In lane A of FIG.15 b the control fusion protein does not have any affinity for TRAP asno TRAP protein can be detected. Lane B demonstrates that the GST-TRIPfusion protein has no affinity for any of the components of bovine serumalbumin (BSA) and is not recognized in the Far-Western. Lanes C and Dshow that GST-TRIP has a dose dependent affinity for TRAP. Lane E is apositive control without glutathione bead extraction, demonstrating thatTRAP can be detected in this Far-Western. These data document theaffinity of TRIP-1 for TRAP and prove that the association does notdepend on post-translational modifications of TRIP-1 as the protein wasproduced in bacteria and that the human sequence of TRIP has affinityfor TRAP in the same way as the molecule from the rat cDNA library.

In order to demonstrate that TRIP-1 and TRAP can interact inside ofcells we utilized a mammalian two hybrid system. In these experiments293T cells were transfected with fusion proteins composed of TRIP-1 witha Gal4-DNA binding domain (DBD) and TRAP with a VP-16-activation domainalong with a luciferase reporter gene. Single transfections with eitherfusion protein showed no increase in reporter activity, however,co-transfection with both fusion proteins showed a 20 fold increase inreporter activity (FIG. 16). Moreover, in a control experiment whereanti-sense TRIP-1 was substituted in the Gal4-DNA binding domain, therewas also no stimulation of the reporter gene. These data demonstratethat TRIP-1 and TRAP can interact with each other in a highly specificmanner in the cytosol of cells. As a further confirmation that TRAP andTRIP-1 interact in the same cell compartment we performed fluorescentlabeling co-localization studies. 293T cells were transfected withTRIP-1 tagged with red fluorescent protein (TRIP-RFP) and TRAP taggedwith a green fluorescent protein (TRAP-GFP). The cells were examined forTRAP-GFP and TRIP-RFP under fluorescence confocal microscopy. The imagesfrom the two wavelengths were digitally superimposed and where red andgreen pixels overlapped we created a merged pseudo-color image. Thisimage demonstrated that at the resolution of the microscope and digitalimage, TRAP and TRIP-1 co-localize inside of cells.

The data in FIG. 17 show that both TGFβ and TRAP can strongly upregulatea reporter gene (P3TP-Lux) that is sensitive to the TGFβ regulatorySmads 2 and 3. The effects of TGFβ and TRAP are additive. As TRIP-1 isknown to interact with the type II TGFβ receptor we investigated whetherTRAP activation of this pathway could be blocked in the presence of adominant negative type II TGFβ receptor expression vector. FIG. 17 alsodemonstrates that in cells that have been co-transfected with a dominantnegative type II TGFβ receptor we can block both TGFβ and TRAPactivation of the pathway. These results were obtained with bothosteoblast cell lines, MG-63 and SaOS2. The data with the MG-63 cell iswhat is shown.

When these experiments were repeated in a Smad4 deficient cell type(SW408 cells), neither TGFβ nor TRAP could activate the reporter gene(FIG. 18). As Smad 4 is a requisite co-factor for Smad 2 and 3signaling, these results are consistent with TRAP working through theSmad pathway. Restoration of Smad4 and TRIP-1 protein in these cells,restores TGFβ and TRAP signaling (FIG. 18). Thus, all of these pieces ofevidence point to the activation of the TGFβ/Smad pathway through theassociation of TRAP with TRIP-1.

TRIP-1 interacted with the type II TGFβ receptor (TGFβRII) when TRAP ispresent in the cytosol. These data are presented in FIG. 19. Theseexperiments utilized a “HIS” tagged TRIP-1 (HIS-TRIP) and a “GST” taggedTRAP (GST-TRAP). Detection of HIS-TRIP was performed with anti-HISantibodies and detection of the TGFβRII and Smad 2 were performed with acommercially available antibodies. For these studies, all cells weretransfected with the TGFβRII. In the first experiment these cells wereco-transfected with HIS-TRIP for 18 hours and then exposed them toexogenously added GST-TRAP for 12 hours. The cells were lysed and thelysate incubated with glutathione beads. The beads were extensivelywashed and the proteins interacting with the beads were analyzed withWestern analysis. FIG. 19 demonstrates that if the cells are not exposedto GST-TRAP (column A) or they are not co-transfected with HIS-TRIP(column C) neither TRIP, TGFβRII nor Smad 2 can be detected. However,when both HIS-TRIP and GST-TRAP are present the complex of the TGFβRII,HIS-TRIP and Smad 2 can be extracted and detected (column B). These dataprovide evidence that in osteoblasts exogenously added TRAP can interactwith cytosolic TRIP and that this complex associates with the type IITGFβ receptor and Smad 2.

As a final test for the ability of osteoblasts to differentiate withinosteoclast lacunae containing TRAP, osteoblasts were cultured oncortical bovine bone wafers on which we had previously createdresorption lacunae with authentic osteoclasts. The lacunae containsubstantial amounts of TRAP. After 7-10 days of culture, onlyosteoblasts residing within the lacunae had differentiated to the pointof producing histochemically detectable alkaline phosphatase (Osteoclastlacunae were created by culturing osteoclasts on cortical bone wafers inthe presence of parathyroid hormone and vitamin D. The osteoclasts wereremoved by gentle scraping. The margin of the lacunae were visible.Osteoblasts were then cultured on these wafers for 10 days and alkalinephosphatase positive cells were identified with histochemical methods.Only the osteoblasts residing within the lacunae demonstrated highlevels of alkaline phosphatase.). This model effectively recapitulatesthe bone remodeling process in vitro and verifies that osteoblasts canexpress a more differentiated phenotype when exposed to molecules withinosteoclast lacunae.

4. Example 4 Tartrate-Resistant Acid Phosphatase Can Induce Apoptosis inMature Osteoblasts a) Abstract

Tartrate-resistant acid phosphatase (TRAP) is an acid hydrolase found athigh concentrations in osteoclasts. TRAP can be recognized byosteoblast-specific genes and that it is one of the anchoring moleculesby which osteoblasts attach to resorption lacunae. TRAP also can induceosteoprogenitor differentiation. In this present paper we describe yetanother function for the enzyme. We show data that indicate that TRAPcan induce apoptosis in aging or scenescent osteoblasts. Morphologicalevidence, DNA fragmentation, biochemical assays for caspases and theirsubstrates and a possible mechanisitic pathway are all consistent withthe conclusion that TRAP can cause cell death and disintegration.

Osteoclast resorption can only occur on a cell-free mineral surface andosteoclast TRAP can be one mechanism by which a the surface may becleaned of lining cells.

b) Materials and Method (1) Cytochemical Staining forScenescence-Activated β-galactosidase

Cells were washed in PBS, fixed for 3-5 min (room temperature) in 2%formaldehyde:0.2% glutaraldehyde. The cells were incubated at 37oC withfresh β-galactosidase stain solution (1 mg of 5-bromo-4-chloro-3-indolylb-D-galactoside per ml, 40 mM citric acid:sodium phosphate, pH 6.0, 5 mMpotas-sium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2mM MgCl2). Staining was evident in 24 h and was maximal in 12-16 h.

(2) In Vitro Cell Senescence

Rat primary calvaria cells were serially passaged by trypsinisation at asplit ratio of 1:2 or 1:4 once they reached confluence. At eachsubculturing, the number of cells was counted using a Coulter counter(Coulter Electronics, UK) and the number of population doublings (PDs)was calculated as log N/log 2, where N is the number of cells in aconfluent layer divided by the initial number of cells seeded.Subculturing continued until the cells reached the end of theirlifespan, which was evident when they failed to become confluent withinfour weeks of culturing. The osteoblastic cells were studied atdifferent time points covering their entire lifespan, and we refer tocells with less than 50% lifespan completed as early-passage youngcells, cells between 60% and 70% lifespan completed are considered to beintermediate-passage middle-aged cells, and cells with more than 90%lifespan completed are considered late-passage senescent cells.

(3) Assays for Apoptosis (a) DNA Fragmentation Assay

After the cultivation of cells in the presence of 5 ug/ml of purifiedTRAP for 36 hrs, cells (5×10⁵) were lysed in a buffer containing 0.5%Triton X-100, 10 mM Tris, pH 7.4,and 10 mM EDTA. After treatment withRNase A and proteinase K, the size of DNA was analyzed by 1.2% agarosegel electrophoresis.

(b) TUNEL Assay

Osteoblastic cells were seeded on chamber slides at a density of 1×10³cells per slide and cultured for 24 hours in the presence of TRAP orcells treated with Etoposide (Sigma, St Louis, Mich.) for 10 minfollowed by 24 h growth was used as positive controls. Apoptosis-inducedDNA strand breaks were detected using the TUNEL (terminaldeoxynucleotidyl transferase-mediated dUTP nick end labeling) technique(Promega, Madsion, Wis.) as described by the manufacturer and analyzedunder a microscope.

(4) RNA Preparation and Quantitative Real-Time Polymerase Chain Reaction(RT-PCR)

Confluent ROS 17/2.8, MC3T3-E1, and primary rat calvaria osteoblasticcultures were grown in standard culture medium. For RNA isolation, cellswere trypsinized, collected in PBS solution and total RNA was extractedby a RNAeasy Mini Kits (Qiagen, Valencia Calif. USA).

cDNA was synthesized from 1 ug of total RNA in a 10-uL reaction mixturecontaining 1 unit reverse transcriptase buffer (5 u=50 mM MgCl₂, 250 mMKCl, 250 mM Tris-HCl (pH 8.3), 50 mM dithiothreitol (DTT), 2.5 mMspermidine), dCTP, dGTP, dATP, and dTTP each at 2 mM, 20 U of RNaseinhibitor, 8-10 U of Superscript II reverse transcriptase 50 pmol ofpoly-dT₁₅ primer (all from Invitrogen, Carlsbad, Calif.). Reaction timeswere at least 1 h at 42° C. After the reverse transcription all thesamples were diluted 1:8 with sterile water and 4 μl of these dilutionswere used for each SYBR.

(5) SYBER Grean Real Time PCR Assay

Each SYBR Green reaction (20 ul total volume) contained 1 ul of dilutedcDNA as template. The final concentration of the reagents were: 1 uMSYBR Green Reaction Buffer, 3 mM MgCl, 1 mM dATP, 1 mM dUTP, 1 mM dCTP,1 mM dGTP, 0.3 uM of each primer, 0.01 U/ml UNG-Enzyme and 0.025 U/mlTaq Gold DNA Polymerase (SYBR Green PCR Core Reagents, PE Biosystems).For all the primer sets, two PCR reactions were carried out with ourstandard SYBR Green protocol with cDNA as template.

The reactions were incubated at 50° C. for 2 min to activate the uracilN-glycosylase (UNG) and then for 5 min at 95° C. to inactivate thisenzyme and activate the Amplitaq Gold polymerase followed by 45 cyclesat 95° C. for 15 sec (denaturation) and at 45° C. for 20 seconds(annealing) and 75° C. for 10 seconds (extension and detection).

c) Results

ROS 17/2.8 cells are a transformed cell line demonstrating many featuresof mature, end stage osteoblasts. Their gene expression profile matchesthe phenotype associated with a fully differentiated bone forming cell.ROS 17/2.8 cells, when exposed, to the osteoclast type V TRAP undergoapoptosis. Moreover, when the cells are stained for alkalinephosphatase, the normally highly expressing control cells shed theiralkaline phosphatase and take on a crenated appearence. The TRAP has nophosphatase activity at the pH tested (7.4) and control phosphatases(ATPase and myokinase) do not show the same effect.

DNA laddering, another hallmark of apoptosis, also occurs in theROS17/2.8 cell line after exposure to TRAP. FIG. 20 demonstrates adose-dependent and time dependent effect of TRAP on DNA. FIG. 20A showsthat after 36 hours a concentration of 5 μg/ml TRAP will induceladdering resulting in fragmentation of the DNA into 180-200 base pairnucleotides. FIG. 20B shows that the laddering will occur at lowerconcentrations (i.e. 0.5 and 1.0 μg/ml) if the cells are exposed to theTRAP for 60 hours.

This induction of apoptosis does not occur in an undifferentiatedpopulation of osteoblasts. Cells freshly prepared from neo natal ratcalvaria as well as the relatively immature MC3T3 osteoblasts are notaffected by TRAP. However, freshly isolated osteoblasts that have beencultured for 14 days under differentiating conditions behave similarlyto the ROS 17/2.8 cells. For these experiments the stage of maturationof the osteoblast cell lines was determined by measuring collagenase IIIlevels and levels of the senescence-associated b-galactosidase enzyme(FIGS. 21A and B). Collagenase III (MP-13) is marker for late stageosteoblast differentiation. It is induced by hormones and factors thatenhance osteoblast development. FIG. 21A shows that early passage ratosteoblasts (day 3) (D3 ROB) and MC3T3-E1 cells have very low levels ofcollagenase III. This level increases as the rat osteoblasts mature (day14) (D14 ROB) and reaches a maximum in ROS 17/2.8 cells (FIG. 21C). FIG.21B demonstrates an increase in the senescence-associatedβ-galactosidase in day 14 osteoblasts, with and without the addition ofthe differentiating agent β-glycerol phosphate. Examination of apoptoticDNA fragmentation in these cells is shown in FIG. 22. In these data,FIG. 22A (MC3T3-E1 cells) and FIG. 22B (D3 ROB) are representative ofosteoblasts at an immature stage. There is no evidence of DNAfragmentation after exposure to TRAP at any concentration in thesecells. However, rat calvarial osteoblasts that have been passaged andcultured for 14 days demonstrate DNA fragmentation after exposure toTRAP at concentrations of 1.0 and 5.0 ug/ml (FIG. 22C).

Verification of apoptosis in day 14 calvarial cells and ROS 17/2.8 cellswas obtained with TUNEL staining. Both scenescent osteoblasts andROS17/2.8 cells are virtually 100% positive for TUNEL staining after a24 hour exposure to TRAP at 5.0 ug/ml. Etoposide, a potentnon-cell-specific inducer of apoptosis, was used a positive control anddemonstrated an effect that was very similar to the TRAP. Untreated ratcalvarial cells and ROS 17/2.8 cells showed no indication of apoptosisby TUNEL staining.

Biochemical confirmation of the effect of TRAP on osteoblast apoptosiswas obtained by assaying levels of caspase-3 and poly ADP-ribosepolymerase (PARP) in these cells lines. Caspase-3 is an enzyme that is akey member of the apoptotic cascade. However, most cells have aconstitutive amount of this enzyme. It is not until the caspase(s) arecleaved that they become activated. Thus, another indicator of a cellsprogression through apoptosis is a measurement of its cleaved caspase 3levels. Similarly, poly ADP-ribose polymerase is a substrate for activecaspase 3 and increased levels of cleave PARP are indicative of anactive caspase.

In FIG. 23 TRAP induces an increase in both cleaved caspase 3 andcleaved PARP in ROS 17/2.8 cells. This occurs after a 36 hour exposureat concentrations of 1.0 and 5.0 ug/ml TRAP. FIGS. 24 and 25 demonstratethat this same effect occurs only in rat calvarial osteoblasts after 14days of culture and not in osteoblasts at 3 days of culture. FIG. 24shows the effect of TRAP on cleavage of PARP by day 14 cells but not day3 cells and FIG. 25 shows cleavage of caspase 3 in day 14 cells treatedwith TRAP. Also in FIG. 25, it was shown that both a purifiedpreparation of TRAP as well as a recombinant fusion protein of GST andTRAP (GST-TRAP) have the same effect on the cells. Usually the regularpeptide hormone EC₅₀ (TGF-b, BMP, PtHrP ) is around umol/L. The maximaldose of TRAP protein used in this paper is also around nmole/L range.

Having confirmed that the TRAP protein was responsible for cell death ofosteoblasts at the late stage of differentiation, differences inphenotype between D3 and D14 primary osteoblasts may protect immatureosteoblasts from TRAP-induced apoptosis were investigated. Becauseosteoprotegrin (OPG) is a known marker for osteoblasts, ELISA assayswere performed to determine whether the two primary osteoblast cellsproduced different levels of OPG (FIG. 26). We found that the level ofOPG in the D14 osteoblasts was the same as that in the D3 osteoblastsprior to exposure to TRAP. However, following TRAP treatment, the levelof OPG increased in the D3 osteoblasts in a dose-dependent manner butdecreased in the D14 osteoblasts.

(1) TRAP Apoptotic Signaling

There are two branches that are important in TGFβ signaling, the Smadpathway and the MAP kinase pathway. As disclosed herein, TRAP isstimulatory for osteoprogenitor cells differentiation through the Smadpathway and addition of TRAP to these cells did not cause apoptosis.However, addition of TRAP to aged osteoblasts induces apoptosis throughthe MAP kinase pathway. Transfection of ROS17/2.8 cells with either adominant negative Ras or a dominant negative Raf, both of which areintermediates in the TGFb/MAP kinase pathway, blocked the apoptoticeffect of TRAP. It was demonstrated that the induction of apoptosis byTRAP in control cells. It was also shown that blockade of Ras signalingprevents apoptosis. The same is true with a blockade of Raf signalingand was shown. These data indicate the MAP kinase branch of the TGFβpathway in mediating the effect of TRAP on aged osteoblasts.

F. Sequences

1. SEQ ID NOS: 1-36 table 4. a) SEQ ID NO: 37 GPC4 nucleic acid genbankaccession number XM_029542 start codon at coding 118-1788   1 ccttctccctccagctccac tcgctagtcc ccgactccgc cagccctcgg cccgctgccg  61 tagcgccgcttcccgtccgg tcccaaaggt gggaacgcgt ccgccccggc ccgcaccatg  121 gcacggttcggcttgcccgc gcttctctgc accctggcag tgctcagcgc cgcgctgctg  181 gctgccgagctcaagtcgaa aagttgctcg gaagtgcgac gtctttacgt gtccaaaggc  241 ttcaacaagaacgatgcccc cctccacgag atcaacggtg atcatttgaa gatctgtccc  301 cagggttctacctgctgctc tcaagagatg gaggagaagt acagcctgca aagtaaagat  361 gatttcaaaagtgtggtcag cgaacagtgc aatcatttgc aagctgtctt tgcttcacgt  421 tacaagaagtttgatgaatt cttcaaagaa ctacttgaaa atgcagagaa atccctgaat  481 gatatgtttgtgaagacata tggccattta tacatgcaaa attctgagct atttaaagat  541 ctcttcgtagagttgaaacg ttactacgtg gtgggaaatg tgaacctgga agaaatgcta  601 aatgacttctgggctcgcct cctggagcgg atgttccgcc tggtgaactc ccagtaccac  661 tttacagatgagtatctgga atgtgtgagc aagtatacgg agcagctgaa gcccttcgga  721 gatgtccctcgcaaattgaa gctccaggtt actcgtgctt ttgtagcagc ccgtactttc  781 gctcaaggcttagcggttgc gggagatgtc gtgagcaagg tctccgtggt aaaccccaca  841 gcccagtgtacccatgccct gttgaagatg atctactgct cccactgccg gggtctcgtg  901 actgtgaagccatgttacaa ctactgctca aacatcatga gaggctgttt ggccaaccaa  961 ggggatctcgattttgaatg gaacaatttc atagatgcta tgctgatggt ggcagagagg 1021 ctagagggtcctttcaacat tgaatcggtc atggatccca tcgatgtgaa gatttctgat 1081 gctattatgaacatgcagga taatagtgtt caagtgtctc agaaggtttt ccagggatgt 1141 ggaccccccaagcccctccc agctggacga atttctcgtt ccatctctga aagtgccttc 1201 agtgctcgcttcagaccaca tcaccccgag gaacgcccaa ccacagcagc tggcactagt 1261 ttggaccgactggttactga tgtcaaggag aaactgaaac aggccaagaa attctggtcc 1321 tcccttccgagcaacgtttg caacgatgag aggatggctg caggaaacgg caatgaggat 1381 gactgttggaatgggaaagg caaaagcagg tacctgtttg cagtgacagg aaatggatta 1441 gccaaccagggcaacaaccc agaggtccag gttgacacca gcaaaccaga catactgatc 1501 cttcgtcaaatcatggctct tcgagtgatg accagcaaga tgaagaatgc atacaatggg 1561 aacgacgtggacttctttga tatcagtgat gaaagtagtg gagaaggaag tggaagtggc 1621 tgtgagtatcagcagtgccc ttcagagttt gactacaatg ccactgacca tgctgggaag 1681 agtgccaatgagaaagccga cagtgctggt gtccgtcctg gggcacaggc ctacctcctc 1741 actgtcttctgcatcttgtt cctggttatg cagagagagt ggagataatt ctcaaactct 1801 gagaaaaagtgttcatcaaa aagttaaaag gcaccagtta tcacttttct accatcctag 1861 tgactttgctttttaaatga atggacaaca atgtacagtt tttactatgt ggccactggt 1921 ttaagaa b)SEQ ID NO: 38 GPC4 peptide genbank accession number XM_029542MARFGLPALLCTLAVLSAALLAAELKSKSCSEVRRLYVSKGFNKNDAPLHEINGDHLKICPQGSTCCSQEMEEKYSLQSKDDFKSVVSEQCNHLQAVFASRYKKFDEFFKELLENAEKSLNDMFVKTYGHLYMQNSELFKDLFVELKRYYVVGNVNLEEMLNDFWARLLERMFRLVNSQYHFTDEYLECVSKYTEQLKPFGDVPRKLKLQVTRAFVAARTFAQGLAVAGDVVSKVSVVNPTAQCTHALLKMIYCSHCRGLVTVKPCYNYCSNIMRGCLANQGDLDFEWNNFIDAMLMVAERLEGPFNIESVMDPIDVKISDAIMNMQDNSVQVSQKVFQGCGPPKPLPAGRISRSISESAFSARFRPHHPEERPTTAAGTSLDRLVTDVKEKLKQAKKFWSSLPSNVCNDERMAAGNGNEDDCWNGKGKSRYLFAVTGNGLANQGNNPEVQVDTSKPDILILRQIMALRVMTSKMKNAYNGNDVDFFDISDESSGEGSGSGCEYQQCPSEFDYNATDHAGKSANEKADSAGVPRGAQAYLLTVFCILFLVMQREWR c) SEQ ID NO: 39 TRIPnucleic acid genbank accession number U36764   1 ggcacgaggt tgcggccttcctcgcgtcac cgccgggatg aagccgatcc  51 tactgcaggg ccatgagcgg tccattacgcagattaagta taaccgcgaa  101 ggagacctcc tctttactgt ggccaaggac cctatcgtcaatgtatggta  151 ctctgtgaat ggtgagaggc tgggcaccta catgggccat accggagctg 201 tgtggtgtgt ggacgctgac tgggacacca agcatgtcct cactggctca  251gctgacaaca gctgtcgtct ctgggactgt gaaacaggaa agcagctggc  301 ccttctcaagaccaattcgg ctgtccggac ctgcggtttt gactttgggg  351 gcaacatcat catgttctccacggacaagc agatgggcta ccagtgcttt  401 gtgagctttt ttgacctgcg ggatccgagccagattgaca acaatgagcc  451 ctacatgaag atcccttgca atgactctaa aatcaccagtgctgtttggg  501 gacccctggg ggagtgcatc atcgctggcc atgagagtgg agagctcaac 551 cagtatagtg ccaagtctgg agaggtgttg gtgaatgtta aggagcactc  601ccggcagatc aacgacatcc agttatccag ggacatgacc atgtttgtga  651 ccgcgtccaaggacaacaca gccaagcttt ttgactccac aactcttgaa  701 catcagaaga ctttccggacagaacgtcct gtcaactcag ctgccctctc  751 ccccaactat gaccatgtgg tcctgggcggtggtcaggaa gccatggatg  801 taaccacaac ctccaccagg attggcaagt ttgaggccaggttcttccat  851 ttggcctttg aagaagagtt tggaagagtc aagggtcact ttggacctat 901 caacagtgtt gccttccatc ctgatggcaa gagctacagc agcggcggcg  951aagatggtta cgtccgtatc cattacttcg acccacagta cttcgaattt 1001 gagtttgaggcttaagaagc tggatctcct gccgggcgtg gtggctcatg 1051 cctgtaatcc caccacttttttttaaggca ggcggatcac ctgaggtcag 1101 gagtttaaga ccagcctgac caacatggagaaactcgtct ctactaaaaa 1151 tacaaaaata caaaaattag ccaggcatgg tggcacacgcctatagtccc 1201 agctactcag gaggctgagg caggagaatc acttgaaccc aggaggcata1251 ggttgcagtg agctgagatc acgtcattgc actccatcct gagccacaag 1301agcaaaactc cgtctcaaaa aaaaaaaa d) SEQ ID NO: 40 TRIP peptide genbankaccession number U36764 MKPILLQGHERSITQIKYNREGDLLFTVAKDPIVNVWYSVNGERLGTYMGHTGAVWCVDADWDTKHVLTGSADNSCRLWDCETGKQLALLKTNSAVRTCGFDFGGNIIMFSTDKQMGYQCFVSFFDLRDPSQIDNNEPYMKIPCNDSKITSAVWGPLGECIIAGHESGELNQYSAKSGEVLVNVKEHSRQINDIQLSRDMTMFVTASKDNTAKLFDSTTLEHQKTFRTERPVNSAALSPNYDHVVLGGGQEAMDVTTTSTRIGKFEARFFHLAFEEEFGRVKGHFGPINSVAFHPDGKSYSSGGEDGYVRIHYFDPQYFEFEFEA e) SEQ ID NO: 41 TRAPnulceic acid XM_032796. Reading frame is 90-1067   1 agggaataaaggctcaggga ccggcagttc tactctagag cccaccagcc tctcagagcc  61 tccggtgactggcctgtgtc tccccctgga tggacatgtg gacggcgctg ctcatcctgc  121 aagccttgttgctaccctcc ctggctgatg gtgccacccc tgccctgcgc tttgtagccg  181 tgggtgactggggaggggtc cccaatgccc cattccacac ggcccgggaa atggccaatg  241 ccaaggagatcgctcggact gtgcagatcc tgggtgcaga cttcatcctg tctctagggg  301 acaatttttacttcactggt gtgcaagaca tcaatgacaa gaggttccag gagacctttg  361 aggacgtattctctgaccgc tcccttcgca aagtgccctg gtacgtgcta gccggaaacc  421 atgaccaccttggcaatgtc tctgcccaga ttgcatactc taagatctcc aagcgctgga  481 acttccccagccctttctac cgcctgcact tcaagatccc acagaccaat gtgtctgtgg  541 ccatttttatgctggacaca gtgacactat gtggcaactc agatgacttc ctcagccagc  601 agcctgagaggccccgagac gtgaagctgg cccgcacaca gctgtcctgg ctcaagaaac  661 agctggcggcggccagggag gactacgtgc tggtggctgg ccactacccc gtgtggtcca  721 tagccgagcacgggcctacc cactgcctgg tcaagcagct acggccactg ctggccacat  781 acggggtcactgcctacctg tgcggccacg atcacaatct gcagtacctg caagatgaga  841 atggcgtgggctacgtgctg agtggggctg ggaatttcat ggacccctca aagcggcacc  901 agcgcaaggtccccaacggc tatctgcgct tccactatgg gactgaagac tcactgggtg  961 gctttgcctatgtggagatc agctccaaag agatgactgt cacttacatc gaggcctcgg 1021 gcaagtccctctttaagacc aggctgccga ggcgagccag gccctgaact cccatgactg 1081 cccagctctgaggcccgatc tccactgttg ggtgggtggg ccctgccggg accctgctca 1141 caggcaggcttttcctccaa cctgtggcgc tgcagcaggg caggaagggg aaacacagct 1201 gatgaactgtggtgccacat gacccttgtg gcacagatgc ccacgtatgt gaaacacaca 1261 tggacatgtgtcccagccac agtgttatgc tctgtggctg gctcaccttt gctgagttcc 1321 ggggtgcaatgggggaggga gggagggaaa gcttcctcct aaatcaagca tctttctgtt 1381 actgatgttcaataaaagaa tagttgccaa ggctg f) SEQ ID NO: 42 TRAP peptideMDMWTALLILQALLLPSLADGATPALRFVAVGDWGGVPNAPFHTAREMANAKEIARTVQILGADFILSLGDNFYFTGVQDINDKRFQETFEDVFSDRSLRKVPWYVLAGNHDHLGNVSAQIAYSKISKRWNFPSPFYRLHFKIPQTNVSVAIFMLDTVTLCGNSDDFLSQQPERPRDVKLARTQLSWLKKQLAAAREDYVLVAGHYPVWSIAEHGPTHCLVKQLRPLLATYGVTAYLCGHDHNLQYLQDENGVGYVLSGAGNFMDPSKRHQRRVPNGYLRFHYGTEDSLGGFAYVEISSKEMTVTYIEASGKSLFKTRLPRRARP g) SEQ ID NO: 43Degenerate of nucleic acid encoding SEQ ID NO: 23 G at position 3 to A5′-ACACCGCTTTCGTATCTGAAGGGTCTGGTGACGGTG-3′ h) SEQ ID NO: 44 Conservativesubstitution in SEQ ID NO: 23 TPLSYLKGLVTI i) SEQ ID NO: 45 degeneratenucleic acid encoding SEQ ID NO: 445′-ACACCGCTTTCGTATCTGAAGGGTCTGGTGACGATA-3′ j) SEQ ID NO: 46 degeneratenucleic acid encoding SEQ ID NO: 445′-ACTCCGCTTTCGTATCTCTAAGGGTCTGGTGACGATA-3′ k) SEQ ID NO: 47 degeneratenucleic acid encoding SEQ ID NO: 38 Atg gcacggttcg gattgcccgc gcttctctgcaccctggcag tgctcagcgc cgcgctgctg gctgccgagc tcaagtcgaa aagttgctcggaagtgcgac gtctttacgt gtccaaaggc ttcaacaaga acgatgcccc cctccacgagatcaacggtg atcatttgaa gatctgtccc cagggttcta cctgctgctc tcaagagatggaggagaagt acagcctgca aagtaaagat gatttcaaaa gtgtggtcag cgaacagtgcaatcatttgc aagctgtctt tgcttcacgt tacaagaagt ttgatgaatt cttcaaagaactacttgaaa atgcagagaa atccctgaat gatatgtttg tgaagacata tggccatttatacatgcaaa attctgagct atttaaagat ctcttcgtag agttgaaacg ttactacgtggtgggaaatg tgaacctgga agaaatgcta aatgacttct gggctcgcct cctggagcggatgttccgcc tggtgaactc ccagtaccac tttacagatg agtatctgga atgtgtgagcaagtatacgg agcagctgaa gcccttcgga gatgtccctc gcaaattgaa gctccaggttactcgtgctt ttgtagcagc ccgtactttc gctcaaggct tagcggttgc gggagatgtcgtgagcaagg tctccgtggt aaaccccaca gcccagtgta cccatgccct gttgaagatgatctactgct cccactgccg gggtctcgtg actgtgaagc catgttacaa ctactgctcaaacatcatga gaggctgttt ggccaaccaa ggggatctcg attttgaatg gaacaatttcatagatgcta tgctgatggt ggcagagagg ctagagggtc ctttcaacat tgaatcggtcatggatccca tcgatgtgaa gatttctgat gctattatga acatgcagga taatagtgttcaagtgtctc agaaggtttt ccagggatgt ggacccccca agcccctccc agctggatgaatttctcgtt ccatctctga aagtgccttc agtgctcgct tcagaccaca tcaccccgaggaacgcccaa ccacagcagc tggcactagt ttggaccgac tggttactga tgtcaaggagaaactgaaac aggccaagaa attctggtcc tcccttccga gcaacgtttg caacgatgagaggatggctg caggaaacgg caatgaggat gactgttgga atgggaaagg caaaagcaggtacctgtttg cagtgacagg aaatggatta gccaaccagg gcaacaaccc agaggtccaggttgacacca gcaaaccaga catactgatc cttcgtcaaa tcatggctct tcgagtgatgaccagcaaga tgaagaatgc atacaatggg aacgacgtgg acttctttga tatcagtgatgaaagtagtg gagaaggaag tggaagtggc tgtgagtatc agcagtgccc ttcagagtttgactacaatg ccactgacca tgctgggaag agtgccaatg agaaagccga cagtgctggtgtccgtcctg gggcacaggc ctacctcctc actgtcttct gcatcttgtt cctggttatgcagagagagt ggagataa l) SEQ ID NO: 48 Conservative substitution in SEQ IDNO: 38 position 5, G to A substitutionMARFALPALLCTLAVLSAALLAAELKSKSCSEVRRLYVSKGFNKNDAPLHEINGDHLKICPQGSTCCSQEMEEKYSLQSKDDFKSVVSEQCNHLQAVFASRYKKFDEFFKELLENAEKSLNDMFVKTYGHLYMQNSELFKDLFVELKRYYVVGNVNLEEMLNDFWARLLERMFRLVNSQYHFTDEYLECVSKYTEQLKPFGDVPRKLKLQVTRAFVAARTFAQGLAVAGDVVSKVSVVNPTAQCTHALLKMIYCSHCRGLVTVKPCYNYCSNIMRGCLANQGDLDFEWNNFIDAMLMYAERLEGPFNIESVMDPIDVKISDAIMNMQDNSVQVSQKVFQGCGPPKPLPAGRISRSISESAFSARFRPHHPEERPTTAAGTSLDRLVTDVKEKLKQAKKFWSSLPSNVCNDERMAAGNGNEDDCWNGKGKSRYLFAVTGNGLANQGNNPEVQVDTSKPDILILRQIMALRVMTSKMKNAYNGNDVDFFDISDESSGEGSGSGCEYQQCPSEFDYNATDHAGKSANEKADSAGVRPGAQAYLLTVFCILFLVMQREWR m) SEQ ID NO: 49degenerate nucleic acid encoding SEQ ID NO: 48 position 5 gcc. Atggcacggttcg ccttgcccgc gcttctctgc accctggcag tgctcagcgc cgcgctgctggctgccgagc tcaagtcgaa aagttgctcg gaagtgcgac gtctttacgt gtccaaaggcttcaacaaga acgatgcccc cctccacgag atcaacggtg atcatttgaa gatctgtccccagggttcta cctgctgctc tcaagagatg gaggagaagt acagcctgca aagtaaagatgatttcaaaa gtgtggtcag cgaacagtgc aatcatttgc aagctgtctt tgcttcacgttacaagaagt ttgatgaatt cttcaaagaa ctacttgaaa atgcagagaa atccctgaatgatatgtttg tgaagacata tggccattta tacatgcaaa attctgagct atttaaagatctcttcgtag agttgaaacg ttactacgtg gtgggaaatg tgaacctgga agaaatgctaaatgacttct gggctcgcct cctggagcgg atgttccgcc tggtgaactc ccagtaccactttacagatg agtatctgga atgtgtgagc aagtatacgg agcagctgaa gcccttcggagatgtccctc gcaaattgaa gctccaggtt actcgtgctt ttgtagcagc ccgtactttcgctcaaggct tagcggttgc gggagatgtc gtgagcaagg tctccgtggt aaaccccacagcccagtgta cccatgccct gttgaagatg atctactgct cccactgccg gggtctcgtgactgtgaagc catgttacaa ctactgctca aacatcatga gaggctgttt ggccaaccaaggggatctcg attttgaatg gaacaatttc atagatgcta tgctgatggt ggcagagaggctagagggtc ctttcaacat tgaatcggtc atggatccca tcgatgtgaa gatttctgatgctattatga acatgcagga taatagtgtt caagtgtctc agaaggtttt ccagggatgtggacccccca agcccctccc agctggacga atttctcgtt ccatctctga aagtgccttcagtgctcgct tcagaccaca tcaccccgag gaacgcccaa ccacagcagc tggcactagtttggaccgac tggttactga tgtcaaggag aaactgaaac aggccaagaa attctggtcctcccttccga gcaacgtttg caacgatgag aggatggctg caggaaacgg caatgaggatgactgttgga atgggaaagg caaaagcagg tacctgtttg cagtgacagg aaatggattagccaaccagg gcaacaaccc agaggtccag gttgacacca gcaaaccaga catactgatccttcgtcaaa tcatggctct tcgagtgatg accagcaaga tgaagaatgc atacaatgggaacgacgtgg acttctttga tatcagtgat gaaagtagtg gagaaggaag tggaagtggctgtgagtatc agcagtgccc ttcagagttt gactacaatg ccactgacca tgctgggaagagtgccaatg agaaagccga cagtgctggt gtccgtcctg gggcacaggc ctacctcctcactgtcttct gcatcttgtt cctggttatg cagagagagt ggagataa n) SEQ ID NO: 50degenerate nucleic acid encoding SEQ ID NO: 48 position 5 gca. Atggcacggttcg cattgcccgc gcttctctgc accctggcag tgctcagcgc cgcgctgctggctgccgagc tcaagtcgaa aagttgctcg gaagtgcgac gtctttacgt gtccaaaggcttcaacaaga acgatgcccc cctccacgag atcaacggtg atcatttgaa gatctgtccccagggttcta cctgctgctc tcaagagatg gaggagaagt acagcctgca aagtaaagatgatttcaaaa gtgtggtcag cgaacagtgc aatcatttgc aagctgtctt tgcttcacgttacaagaagt ttgatgaatt cttcaaagaa ctacttgaaa atgcagagaa atccctgaatgatatgtttg tgaagacata tggccattta tacatgcaaa attctgagct atttaaagatctcttcgtag agttgaaacg ttactacgtg gtgggaaatg tgaacctgga agaaatgctaaatgacttct gggctcgcct cctggagcgg atgttccgcc tggtgaactc ccagtaccactttacagatg agtatctgga atgtgtgagc aagtatacgg agcagctgaa gcccttcggagatgtccctc gcaaattgaa gctccaggtt actcgtgctt ttgtagcagc ccgtactttcgctcaaggct tagcggttgc gggagatgtc gtgagcaagg tctccgtggt aaaccccacagcccagtgta cccatgccct gttgaagatg atctactgct cccactgccg gggtctcgtgactgtgaagc catgttacaa ctactgctca aacatcatga gaggctgttt ggccaaccaaggggatctcg attttgaatg gaacaatttc atagatgcta tgctgatggt ggcagagaggctagagggtc ctttcaacat tgaatcggtc atggatccca tcgatgtgaa gatttctgatgctattatga acatgcagga taatagtgtt caagtgtctc agaaggtttt ccagggatgtggacccccca agcccctccc agctggacga atttctcgtt ccatctctga aagtgccttcagtgctcgct tcagaccaca tcaccccgag gaacgcccaa ccacagcagc tggcactagtttggaccgac tggttactga tgtcaaggag aaactgaaac aggccaagaa attctggtcctcccttccga gcaacgtttg caacgatgag aggatggctg caggaaacgg caatgaggatgactgttgga atgggaaagg caaaagcagg tacctgtttg cagtgacagg aaatggattagccaaccagg gcaacaaccc agaggtccag gttgacacca gcaaaccaga catactgatccttcgtcaaa tcatggctct tcgagtgatg accagcaaga tgaagaatgc atacaatgggaacgacgtgg acttctttga tatcagtgat gaaagtagtg gagaaggaag tggaagtggctgtgagtatc agcagtgccc ttcagagttt gactacaatg ccactgacca tgctgggaagagtgccaatg agaaagccga cagtgctggt gtccgtcctg gggcacaggc ctacctcctcactgtcttct gcatcttgtt cctggttatg cagagagagt ggagataa o) SEQ ID NO:51-78, sequences related to linkers PRFKIIGG, SEQ ID NO: 51 PRFRIIGG,SEQ ID NO: 52 SSRHRRALD, SEQ ID NO: 53 RKSSIIIRMRDVVL, SEQ ID NO: 54SSSFDKGKYKKGDDA, SEQ ID NO: 55 SSSFDKGKYKRGDDA, SEQ ID NO: 56 IEGR, SEQID NO: 57 IDGR, SEQ ID NO: 58 GGSIDGR, SEQ ID NO: 59 PLGLWA, SEQ ID NO:60 GPQGIAGQ, SEQ ID NO: 61 GPQGLLGA, SEQ ID NO: 62 GIAQQ, SEQ ID NO: 63QPLGIAGI, SEQ ID NO: 64 GPEGLRVG, SEQ ID NO: 65 YGAGLGVV, SEQ ID NO: 66AGLGVVER, SEQ ID NO: 67 AGLGISST, SEQ ID NO: 68 EPQALAMS, SEQ ID NO: 69QALAMSAI, SEQ ID NO: 70 AAYHLVSQ, SEQ ID NO: 71 MDAFLESS, SEQ ID NO: 72ESLPVVAV, SEQ ID NO: 73 SAPAVESE, SEQ ID NO: 74 DVAQFVLT, SEQ ID NO: 75VAQFVLTE, SEQ ID NO: 76 AQFVLTEG, SEQ ID NO: 77 PVQPIGPQ, SEQ ID NO: 78

1. An implant comprising a peptide comprising the sequence set forth inSEQ ID NO:42, or conservative variant or fragment thereof:
 2. An implantcomprising a peptide comprising a sequence having at least 80% identityto the sequence set forth in SEQ ID NO:42, or conservative variant orfragment thereof.
 3. The implant of claim 1, wherein the implant is adental implant.
 4. The implant of claim 1, wherein the peptide can becleaved from the implant in the environment of the bone.
 5. An isolatedcomposition comprising a peptide that binds TRAP, wherein the moleculedoes not have a sequence set forth in SEQ ID NO:38 or SEQ ID NO:40. 6.The composition of claim 5, wherein the composition binds TRAP with a Kdless than or equal to 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10¹⁰ ⁻M,10⁻¹¹ M, or 10⁻¹² M.
 7. The composition of claim 5, wherein thecomposition comprises a peptide selected from the sequences set forth inSEQ ID NO:19-36.
 8. A composition comprising a peptide set forth in SEQID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, or SEQ ID NO:36, or conserved variant or fragmentthereof, wherein the composition binds TRAP, wherein the peptide is notSEQ ID NO:38 or SEQ ID NO:40.
 9. A composition comprising at least 80%identity to a peptide set forth in SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or SEQ IDNO:36, or conserved variant or fragment thereof, wherein the compositionbinds TRAP, wherein the peptide is not set forth in SEQ ID NO:38 or SEQID NO:40.
 10. The composition of claim 8, wherein the composition bindsTRAP with a Kd less than or equal to 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M,10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M.
 11. A process for producing apeptide that binds TRAP comprising expressing the peptide from a nucleicacid or chemically synthesizing the peptide, wherein the peptide is setforth in claim
 8. 12. A process for producing a cell comprising acomposition that binds TRAP comprising administering the peptide setforth in claim 8 to the cell.
 13. A process for producing an animalcomprising a composition that binds TRAP, comprising administering thepeptide set forth in claim 8 to the animal.
 14. A process for producingan animal comprising a composition that binds TRAP, comprisingadministering the cell set forth in claim 12 to the animal.
 15. Aprocess for producing an animal comprising a composition that bindsTRAP, comprising administering the implant set forth in claim 1 to theanimal.
 16. The process of claim 13, wherein the animal is a mammal. 17.The process of claims 16, wherein the mammal is a mouse, rat, rabbit,cow, sheep, pig, or primate.
 18. An isolated composition comprising amolecule that binds TRAP, wherein the molecule does not have a sequenceset forth in SEQ ID NO:38 or SEQ ID NO:40, and wherein the compositionbinds amino acids 252-263 of SEQ ID NO:38 or a conserved variant orfragment thereof.
 19. The composition of claim 18, wherein thecomposition binds amino acids 259-263 of SEQ ID NO:38 or a conservedvariant or fragment thereof.
 20. A method of regulating bone formationcomprising administering a composition, wherein the composition bindsGPC4.
 21. The method of claim 20, wherein the composition is a peptidehaving at least 80% identity to SEQ ID NO:42, or a conservative variantor fragment thereof.
 22. The method of claim 21, wherein the peptide isset forth in SEQ ID NO:42.
 23. A method of regulating bone formationcomprising administering a composition, wherein the composition bindswith TRIP.
 24. The method of claim 23, wherein the composition is apeptide having at least 80% identity to SEQ ID NO:42, or a conservativevariant or fragment thereof.
 25. The method of claim 24, wherein thepeptide is set forth in SEQ ID NO:42.
 26. The method of claim 20,wherein regulating bone formation comprises decreasing bone formation.27. The method of claim 26, wherein decreasing bone formation comprisesinhibiting osteoblast binding to osteoclast lacunae.
 28. The method ofclaim 20, wherein regulating bone formation comprises increasing boneformation.
 29. The method of claim 28, wherein increasing bone formationcomprises increasing osteoblast binding to osteoclast lacunae.
 30. Themethod of claim 29, wherein increasing bone formation comprisesincreasing osteoblast differentiation.
 31. The method of claim 20,wherein the composition is administered systemically.
 32. The method ofclaim 31, wherein the composition is administered intravenously orintra-arterially.
 33. The method of claim 20, wherein the composition isadministered by implanting on bone.
 34. The method of claim 20, whereinthe administering the composition occurs in bone cell culture.
 35. Themethod of claims 34, wherein the bone cell culture comprises osteoblastcells or osteoclast cells.
 36. The method of claim 20, wherein thecomposition is administered to a patient having a bone related disorder.37. The method of claim 36, wherein the disorder is type Ipostmenopausal osteoporosis; type II age-related osteoporosis; maleosteoporosis; secondary osteoporosis due to steroid or pharmaceuticaluse, renal osteodystrophy, renal stones, juvenile idiopathicosteoporosis, hyperparathyroidism, hyperthyroidism, hypercalcemia's,Fanconi syndrome, sarcoidosis, diabetes, osteomalacia, VDRR, VDDR, andnutritional rickets, hypervitaminosis A and D, Paget's Disease,osteopetrosis, skeletal tumors, rheumatoid and osteo arthritis,osteogensis imperfecta, chondrodystrophies or sclerosing bonedysplasias.
 38. The method of claim 36, wherein the disorder comprisesheterotopic bone formation, osteophyte formation, diffuse idiopathicskeletal hyperostosis (DISH), or myositis ossificans progressiva (MOP).39. A method of anchoring an implant comprising inserting an implantcomprising TRAP or a conserved variant or fragment of TRAP.
 40. Themethod of claim 39, wherein the implant is the implant of claims 1-4.41. A nucleic acid molecule comprising a sequence set forth in SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, or degenerate variant or fragment thereof.
 42. Anucleic acid molecule comprising a sequence having at least 80% identityto a sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or degeneratevariant or fragment thereof.
 43. A nucleic acid molecule comprising asequence that encodes a peptide set forth in SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,SEQ, or SEQ ID NO:36, or a conserved variant or fragment thereof,wherein the peptide sequence binds TRAP.
 44. A nucleic acid moleculecomprising a sequence that encodes a peptide having at least 80%identity to a peptide set forth in SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, or a conserved variant or fragment thereof, wherein the peptidesequence binds TRAP.
 45. The nucleic acid of claim 41, wherein thepeptide encoded by the nucleic acid binds TRAP with a Kd less than orequal to 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or10⁻¹² M.
 46. A vector comprising the nucleic acid of claim
 41. 47. Thevector of claim 46, wherein the nucleic acid is under the control of acell specific promoter.
 48. The vector of claim 47, wherein the promoteris a type I collagen promoter, an alkaline phosphatase promoter, anosteonectin promoter, an osteocalcin promoter, or a cbfa1 promoter. 49.A cell comprising the nucleic acid of claim
 41. 50. The cell of claim48, wherein the cell is a fibroblast cell, a cartilage cell, a bonecell, bone marrow cell, a stem cell, an adipocyte cell, or an osteoblastcell.
 51. A pharmaceutical composition comprising the nucleic acid ofclaim
 41. 52. A pharmaceutical composition comprising the peptide ofclaim
 5. 53. A pharmaceutical composition comprising the cell of claim48.